Anti-sez6 antibody drug conjugates and methods of use

ABSTRACT

Provided are novel anti-SEZ6 antibodies and antibody drug conjugates, and methods of using such anti-SEZ6 antibodies and antibody drug conjugates to treat cancer.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/678,061, filed May 30, 2018, the contentsof which are incorporated herein in its entirety by reference thereto.

2. REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 30, 2019, isnamed 381493-703WO(167862)_SL.txt and is 28,992 bytes in size.

3. TECHNICAL BACKGROUND

The present application pertains to a novel anti-SEZ6 antibody drugconjugate, components thereof and compositions comprising the same forthe treatment, diagnosis or prophylaxis of cancer and any recurrence ormetastasis thereof.

4. BACKGROUND OF THE INVENTION

Differentiation and proliferation of stem cells and progenitor cells arenormal ongoing processes that act in concert to support tissue growthduring organogenesis, cell repair and cell replacement. The system istightly regulated to ensure that only appropriate signals are generatedbased on the needs of the organism. Cell proliferation anddifferentiation normally occur only as necessary for the replacement ofdamaged or dying cells or for growth. However, disruption of theseprocesses can be triggered by many factors including the under- oroverabundance of various signaling chemicals, the presence of alteredmicroenvironments, genetic mutations or a combination thereof.Disruption of normal cellular proliferation and/or differentiation canlead to various disorders including proliferative diseases such ascancer.

Conventional therapeutic treatments for cancer include chemotherapy,radiotherapy and immunotherapy. Often these treatments are ineffectiveand surgical resection may not provide a viable clinical alternative.Limitations in the current standard of care are particularly evident inthose cases where patients undergo first line treatments andsubsequently relapse. In such cases refractory tumors, often aggressiveand incurable, frequently arise. The overall survival rates for manytumors have remained largely unchanged over the years due, at least inpart, to the failure of existing therapies to prevent relapse, tumorrecurrence and metastasis. There remains therefore a great need todevelop more targeted and potent therapies for proliferative disorders.The current invention addresses this need.

5. SUMMARY OF THE INVENTION

In a broad aspect the present invention provides an antibody drugconjugate, or compositions thereof, which specifically binds to humanSEZ6 determinants. In certain embodiments the SEZ6 determinant is a SEZ6protein expressed on tumor cells while in other embodiments the SEZ6determinant is expressed on tumor initiating cells. In a preferredembodiment the SEZ6 antibody drug conjugate will comprise:

wherein Ab comprises an anti-SEZ6 antibody having a heavy chain of SEQID NO:3 and a light chain of SEQ ID NO:4 and wherein n is 2. For thepurposes of the instant disclosure this antibody drug conjugate shall betermed “hSEZ6-1.ss1 ADC1” unless otherwise indicated.

In selected embodiments the present invention comprises an antibodycomprising a heavy chain of SEQ ID NO:3 and a light chain of SEQ IDNO:4. In certain aspects the invention comprises a nucleic acid encodinga heavy chain (SEQ ID NO:3) or light chain (SEQ ID NO:4) of theanti-SEZ6 antibody of the invention or a fragment thereof. In otherembodiments the invention comprises a vector comprising one or more ofthe above described nucleic acids or a host cell comprising said nucleicacids or vectors.

In yet another embodiment the invention comprises a calicheamicin druglinker, or a pharmaceutically acceptable salt or solvate thereof,comprising the structure set forth in Formula II.

As set forth above the present invention provides an anti-SEZ6 antibodydrug conjugate wherein the antibody is conjugated to one or morenon-cleavable calicheamicin payloads of Formula II. Further provided arepharmaceutical compositions comprising an anti-SEZ6 ADC as disclosedherein. In certain embodiments the compositions will comprise a selecteddrug-antibody ratio (DAR) where the predominant ADC species (e.g.,comprising 2 calicheamicin warheads) comprises greater than about 50%,greater than about 60%, greater than about 70%, greater than about 80%,greater than about 90% or greater than about 95% of the species present.Preferably the drug loading of the predominant species will be 2.

Another aspect of the invention is a method of treating cancercomprising administering an antibody drug conjugate or a pharmaceuticalcomposition such as those described herein to a subject in need thereof.In certain embodiments the subject will be suffering from lung cancerand, in selected embodiments, small cell lung cancer (SCLC).

In other embodiments the disclosed ADC will comprise a safety margin(derived as described herein) greater than 6. In other embodiments thesafety margin will be greater than 7 and in yet other embodiments thesafety margin will be greater than 8 or greater than 9. In still otherembodiments the safety margin will be about 10.

In still another embodiment the invention comprises a method of reducingtumor initiating cells in a tumor cell population, wherein the methodcomprises contacting (e.g. in vitro or in vivo) a tumor initiating cellpopulation with an antibody drug conjugate as described herein wherebythe frequency of the tumor initiating cells is reduced.

In one aspect, the invention comprises a method of delivering acytotoxin to a cell comprising contacting the cell with the disclosedantibody drug conjugate.

In another aspect the present invention also provides kits or devicesand associated methods that are useful in the treatment of lung cancer.To this end the present invention preferably provides an article ofmanufacture useful for treating lung cancer comprising a receptaclecontaining the SEZ6 ADC and instructional materials for using the ADC totreat lung cancer (e.g., small cell lung cancer) or provide a dosingregimen for the same. In other embodiments the disclosed kits willcomprise instructions, labels, inserts, readers or the like indicatingthat the kit or device is used for the treatment of lung cancer orprovide a dosing regimen for the same.

The foregoing is a summary and thus contains, by necessity,simplifications, generalizations, and omissions of detail; consequently,those skilled in the art will appreciate that the summary isillustrative only and is not intended to be in any way limiting. Otheraspects, features, and advantages of the methods, compositions and/ordevices and/or other subject matter described herein will becomeapparent in the teachings set forth herein. The summary is provided tointroduce a selection of concepts in a simplified form that are furtherdescribed below in the Detailed Description.

6. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of hSEZ6-1.ss1 ADC1;

FIG. 2 provides binding curves demonstrating that a SEZ6 antibodycomprising the S60N mutation has an affinity profile that issubstantially the same as a SEZ6 antibody without the S60N mutation;

FIG. 3 demonstrates that the disclosed SEZ6 ADCs effectively kill cellsexpressing hSEZ6 in a dose dependent manner while not depleting naïvecontrol cells that do not express hSEZ6;

FIGS. 4A and 4B depict the ability of the disclosed SEZ6 ADCs to retardsmall cell lung cancer tumor growth in immunocompromised mice implantedwith the SCLC LU 95 (FIG. 4A) and SCLC LU 149 (FIG. 4B); and

FIGS. 5A-5D demonstrate that SCLC tumors are particularly susceptible totreatment with ADCs comprising a non-cleavable calicheamicin linker drugwherein FIGS. 5A and 5B show, respectively, relative expression levelsof SEZ6 and a positive control antigen in various tumor cell lines, FIG.5C shows that a PBD toxin uniformly kills different type of cancer cellsand FIG. 5D shows that calicheamicin is particularly active in killingSCLC cells.

7. DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are non-limiting, illustrative embodiments of theinvention that exemplify the principles thereof. Any section headingsused herein are for organizational purposes only and are not to beconstrued as limiting the subject matter described. For the purposes ofthe instant disclosure all identifying sequence accession numbers may befound in the NCBI Reference Sequence (RefSeq) database and/or the NCBIGenBank® archival sequence database unless otherwise noted.

The present invention provides novel anti-SEZ6 ADCs and componentsthereof (including the hSEZ6-1.ss1 antibody and the calicheamicin druglinker of Formula II) comprising a site-specific anti-SEZ6 antibodytargeting agent and calicheamicin cytotoxic payload. As discussed inmore detail below and set forth in the appended Examples, the disclosedanti-SEZ6 ADC is particularly efficacious in suppressing tumor growthwhen compared with other anti-SEZ6 ADCs.

The ability to effectively reduce or eliminate tumor and/or cancer stemcells through use of the SEZ6 ADC disclosed herein is due to therelative lack of off-site toxicity which allows for increased dosing.This, in turn provides for higher calicheamicin levels at the tumor sitethan other calicheamicin ADCs and results in increased cell killing. Asshown in the appended Examples the increased toxin concentration at thetumor-site provides for extended tumor suppression in immunocompromisedmice. Thus, the SEZ6 ADC disclosed herein will likely exhibit afavorable therapeutic index and may be used in the treatment and/orprevention of selected proliferative disorders such as small cell lungcancer or progression or recurrence thereof.

SEZ6 Cancer Stem Cells

It has been postulated that SCLC is bronchogenic in origin, arising inpart from pulmonary neuroendocrine cells (Galluzzo and Bocchetta, 2011;PMID: 21504320). Whatever the cellular source of origin for thesetumors, it is clear that they show a poorly differentiated endocrinephenotype, often are highly proliferative and aggressive, and frequentlyover-express neural proteins. The resultant elevation of neuralexpression markers in these tumors that otherwise may be primarilyrestricted to the nervous system or show limited expression duringdevelopment, of which SEZ6 may be an exemplar, may therefore offer aunique therapeutic target for tumors with the neuroendocrine phenotype.

SEZ6 expression is associated with various tumorigenic cellsubpopulations in a manner which renders them susceptible to treatmentas set forth herein. In this regard the invention provides hSEZ6-1.ss1ADC1 which may be particularly useful for targeting tumorigenic cellsthereby facilitating the treatment, management and/or prevention ofcancer. Whether by inhibition or elimination of the tumorigenic cells,modification of their potential (for example, by induced differentiationor niche disruption) or otherwise interfering with the ability oftumorigenic cells to influence the tumor environment or other cells, thepresent invention allows for more effective treatment of cancer byinhibiting tumorigenesis, tumor maintenance and/or metastasis andrecurrence. It will further be appreciated that the same characteristicsof the disclosed antibodies make them particularly effective at treatingrecurrent tumors which have proved resistant or refractory to standardtreatment regimens.

Methods that can be used to assess a reduction in the frequency oftumorigenic cells include, but are not limited to, cytometric orimmunohistochemical analysis, preferably by in vitro or in vivo limitingdilution analysis (Dylla et al. 2008, PMID: PMC2413402 and Hoey et al.2009, PMID: 19664991).

The ability of the antibodies of the current invention to reduce thefrequency of tumorigenic cells can therefore be determined using thetechniques and markers known in the art. In some instances, theanti-SEZ6 ADCs may reduce the frequency of tumorigenic cells by 10%,15%, 20%, 25%, 30% or even by 35%. In other embodiments, the reductionin frequency of tumorigenic cells may be in the order of 40%, 45%, 50%,55%, 60% or 65%. In certain embodiments, the disclosed compounds mayreduce the frequency of tumorigenic cells by 70%, 75%, 80%, 85%, 90% oreven 95%. It will be appreciated that any reduction of the frequency oftumorigenic cells is likely to result in a corresponding reduction inthe tumorigenicity, persistence, recurrence and aggressiveness of theneoplasia.

Antibody Structure

Antibodies and variants and derivatives thereof, including acceptednomenclature and numbering systems, have been extensively described, forexample, in Abbas et al. (2010), Cellular and Molecular Immunology(6^(th) Ed.), W.B. Saunders Company; or Murphey et al. (2011), Janeway'sImmunobiology (8^(th) Ed.), Garland Science.

An “antibody” or “intact antibody” typically refers to a Y-shapedtetrameric protein comprising two heavy (H) and two light (L)polypeptide chains held together by covalent disulfide bonds andnon-covalent interactions. Each light chain is composed of one variabledomain (VL) and one constant domain (CL). Each heavy chain comprises onevariable domain (VH) and a constant region, which in the case of IgG,IgA, and IgD antibodies, comprises three domains termed CH1, CH2, andCH3 (IgM and IgE have a fourth domain, CH4). In IgG, IgA, and IgDclasses the CH1 and CH2 domains are separated by a flexible hingeregion, which is a proline and cysteine rich segment of variable length(from about 10 to about 60 amino acids in various IgG subclasses). Thevariable domains in both the light and heavy chains are joined to theconstant domains by a “J” region of about 12 or more amino acids and theheavy chain also has a “D” region of about 10 additional amino acids.Each class of antibody further comprises inter-chain and intra-chaindisulfide bonds formed by paired cysteine residues.

As used herein the term “antibody” specifically includes humanized IgG1monoclonal antibodies comprising kappa (κ) light chains.

The variable domains of antibodies show considerable variation in aminoacid composition from one antibody to another and are primarilyresponsible for antigen recognition and binding. Variable regions ofeach light/heavy chain pair form the antibody binding site such that anintact IgG antibody has two binding sites (i.e. it is bivalent). VH andVL domains comprise three regions of extreme variability, which aretermed hypervariable regions, or more commonly,complementarity-determining regions (CDRs), framed and separated by fourless variable regions known as framework regions (FRs). Non-covalentassociation between the VH and the VL region forms the Fv fragment (for“fragment variable”) which contains one of the two antigen-binding sitesof the antibody.

As used herein, the assignment of amino acids to each domain, frameworkregion and CDR will be in accordance with one of the schemes provided byKabat et al. (1991) Sequences of Proteins of Immunological Interest(5^(th) Ed.), US Dept. of Health and Human Services, PHS, NIH, NIHPublication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia etal., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; orDubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCHVerlag GmbH and Co or AbM (Oxford Molecular/MSI Pharmacopia) unlessotherwise noted. As is well known in the art variable region residuenumbering is typically as set forth in Chothia or Kabat. Amino acidresidues which comprise CDRs as defined by Kabat, Chothia, MacCallum(also known as Contact) and AbM as obtained from the Abysis websitedatabase (infra.) are set out below in Table 1. Note that MacCallum usesthe Chothia numbering system.

TABLE 1 Rabat Chothia MacCallum AbM VH CDR1 31-35 26-32 30-35 26-35 VHCDR2 50-65 52-56 47-58 50-58 VH CDR3  95-102  95-102  93-101  95-102 VLCDR1 24-34 24-34 30-36 24-34 VL CDR2 50-56 50-56 46-55 50-56 VL CDR389-97 89-97 89-96 89-97

Variable regions and CDRs in an antibody sequence can be identifiedaccording to general rules that have been developed in the art (e.g., asset forth above) or by aligning the sequences against a database ofknown variable regions. Methods for identifying these regions aredescribed in Kontermann and Dubel, eds., Antibody Engineering, Springer,New York, N.Y., 2001 and Dinarello et al., Current Protocols inImmunology, John Wiley and Sons Inc., Hoboken, N.J., 2000. Exemplarydatabases of antibody sequences are described in, and can be accessedthrough, the “Abysis” website at www.bioinf.org.uk/abs (maintained by A.C. Martin in the Department of Biochemistry & Molecular BiologyUniversity College London, London, England) and the VBASE2 website atwww.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33(Database issue): D671-D674 (2005).

Preferably sequences are analyzed using the Abysis database, whichintegrates sequence data from Kabat, IMGT and the Protein Data Bank(PDB) with structural data from the PDB. See Dr. Andrew C. R. Martin'sbook chapter Protein Sequence and Structure Analysis of AntibodyVariable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S.and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13:978-3540413547, also available on the website bioinforg.uk/abs). TheAbysis database website further includes general rules that have beendeveloped for identifying CDRs which can be used in accordance with theteachings herein. Unless otherwise indicated, all CDRs set forth hereinare derived according to the Abysis database website as per Kabat et al.

For heavy chain constant region amino acid positions discussed in theinvention, numbering is according to the Eu index first described inEdelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1): 78-85 describingthe amino acid sequence of the myeloma protein Eu, which reportedly wasthe first human IgG1 sequenced. The Eu index of Edelman is also setforth in Kabat et al., 1991 (supra.). Thus, the terms “Eu index as setforth in Kabat” or “Eu index of Kabat” or “Eu index” or “Eu numbering”in the context of the heavy chain refers to the residue numbering systembased on the human IgG1 Eu antibody of Edelman et al. as set forth inKabat et al., 1991 (supra.) The numbering system used for the lightchain constant region amino acid sequence is similarly set forth inKabat et al., (supra.).

Those of skill in the art will appreciate that the heavy and light chainconstant region sequences of the hSEZ6-1.ss1 antibody have beenengineered as disclosed herein to provide unpaired cysteines. Theseconstant regions are then operably associated with the disclosed heavyand light chain variable regions using standard molecular biologytechniques to provide the full-length antibody chains that areincorporated in the disclosed hSEZ6-1.ss1.

Antibody Generation and Production

The humanized antibody hSEZ6-1.ss1 is produced as set forth in Examples1 and 2 appended hereto and the resulting amino acid sequences of thefull length heavy chain and full length light chain are set forthimmediately below as SEQ ID NO:3 and SEQ ID NO:4. As discussed below theheavy chain of the humanized antibody comprises a S60N mutation designedto inactivate a glycosylation site and a C220S mutation (numberedaccording to the EU index of Kabat) that provides the free cysteine onthe light chain. Note that in the heavy chain the S60N and the C220Smutations are underlined while the resulting free cysteine at position214 is underlined in the light chain. The variable region of both chainsis depicted in bold type.

(SEQ ID NO: 3) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSSWINWVRQMPGKGLEWMGRIYPGEGDTNY N GNFEGQVTISADKSISTAYLQWSSLKASDTAMYYCTRGLVMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 4)EIVLTQSPATLSLSPGERATLSCRASQSVDYNGISYMHWYQQKPGQAPRLLIYAASNVQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSIEDPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

In addition, compatible nucleic acid sequences encoding theaforementioned full length heavy and light chains are set forthimmediately below as SEQ ID NO:5 and SEQ ID NO:6 respectively.

HEAVY CHAIN NUCLEIC ACID SEQUENCE (with introns) (SEQ ID NO: 5)gaagtccaactcgtccaatccggtgccgaagtgaaaaagcctggggaatccctgaagatcagctgcaagggatccggttactcgttcacctcctcctggattaactgggtccggcagatgcccggaaagggactggagtggatgggcagaatctatccgggcgaaggggacactaattacaacggaaacttcgagggccaggtcaccatttcggccgataagagcatctcaaccgcgtacttgcagtggtcaagcctgaaggcttccgacaccgccatgtactactgtactcgcggccttgtgatggactactggggacagggaactctcgtgaccgtgtcgtccgcctctaccaagggcccttccgtgttccctctggccccctcgagcaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgagccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttggtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggaggcctctgcccgccccactcatgctcagggagagggtcttctggctttttccccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctctgcagagcccaaatctagtgacaaaactcacacatgcccaccgtgcccaggtaagccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctccatctcttcctcagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaaggtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt LIGHT CHAIN NUCLEIC ACID SEQUENCE(SEQ ID NO: 6) gaaatcgtgttgacccagtcccccgctaccctgtcactgagccccggagaacgcgcgactctgtcctgccgggcatcccagtccgtggactacaacggaatctcctacatgcactggtatcagcaaaagccaggccaagccccgagactgctcatctacgccgcctcgaacgtgcagagcggtattccggcgcggttctccggctcgggcagcggaaccgattttaccctcactatctcgtcacttgaacctgaggacttcgccgtgtactactgccagcagtccattgaggacccgcctactttcggggggggaaccaaagtcgagatcaagcggactgtggctgcaccaagtgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagct tcaacaggggagagtgt

The SEZ6 antibody component of the instant invention has been engineeredto facilitate efficient conjugation of the calicheamicin toxin. In thisregard antibody drug conjugate (ADC) preparations of the inventioncomprise a relatively homogenous population of ADC molecules both interms of the position of the cytotoxin on the antibody and the drug toantibody ratio (DAR). Based on the instant disclosure one skilled in theart could readily fabricate the disclosed site-specific engineeredconstruct comprising SEQ ID NOS:3 and 4. As used herein a “site-specificantibody” or “site-specific construct” means an antibody wherein atleast one amino acid in either the heavy or light chain is deleted,altered or substituted (preferably with another amino acid) to provideat least one free cysteine. Similarly, a “site-specific conjugate” shallbe held to mean an ADC comprising a site-specific antibody and at leastone cytotoxin conjugated to the unpaired or free cysteine(s). In thepresent invention the cysteine at position 220 of the heavy chain (Eunumbering of Kabat) has been mutated to a serine thereby disrupting thedisulfide bridge that naturally forms with the terminal cysteine atposition 214 of the kappa light chain constant region. This renders thecysteine at position 214 a free or unpaired cysteine in accordance withthe teachings herein that is then conjugated to the disclosedcalicheamicin drug linker.

As used herein, the terms “free cysteine” or “unpaired cysteine” may beused interchangeably unless otherwise dictated by context and shall meana cysteine (or thiol containing) constituent (e.g., a cysteine residue)of an antibody, whether naturally present or specifically incorporatedin a selected residue position using molecular engineering techniques,that is not part of a naturally occurring (or “native”) disulfide bondunder physiological conditions. In selected embodiments the freecysteine may comprise a naturally occurring cysteine whose nativeinterchain or intrachain disulfide bridge partner has been substituted,eliminated or otherwise altered to disrupt the naturally occurringdisulfide bridge under physiological conditions thereby rendering theunpaired cysteine suitable for site-specific conjugation. It will beappreciated that, prior to conjugation, free or unpaired cysteines maybe present as a thiol (reduced cysteine), as a capped cysteine(oxidized) or as part of a non-native intra- or intermolecular disulfidebond (oxidized) with another cysteine or thiol group on the same ordifferent molecule depending on the oxidation state of the system. Asdiscussed in more detail below, mild reduction of the appropriatelyengineered antibody construct will provide thiols available forsite-specific conjugation. Accordingly, in particularly preferredembodiments the free or unpaired cysteines will be subject to selectivereduction and subsequent conjugation to provide homogenous DARcompositions. No longer “free” or “unpaired” such residue positions maybe termed “engineered sites of conjugation” once they are covalentlybound to the drug linker.

Antibodies and fragments thereof may be produced or modified usinggenetic material obtained from antibody producing cells and recombinanttechnology (see, for example; Dubel and Reichert (Eds.) (2014) Handbookof Therapeutic Antibodies, 2^(nd) Edition, Wiley-Blackwell GmbH;Sambrook and Russell (Eds.) (2000) Molecular Cloning: A LaboratoryManual (3^(rd) Ed.), NY, Cold Spring Harbor Laboratory Press; Ausubel etal. (2002) Short Protocols in Molecular Biology: A Compendium of Methodsfrom Current Protocols in Molecular Biology, Wiley, John & Sons, Inc.;and U.S. Pat. No. 7,709,611).

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibody of the invention. The nucleic acids may be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or renderedsubstantially pure when separated from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. A nucleic acid of the invention can be, for example, DNA(e.g. genomic DNA, cDNA), RNA and artificial variants thereof (e.g.,peptide nucleic acids), whether single-stranded or double-stranded orRNA, RNA and may or may not contain introns. In selected embodiments thenucleic acid is a cDNA molecule.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared as described in the Examples below), cDNAs encodingthe light and heavy chains of the antibody can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid molecules encoding the antibody can berecovered from the library.

DNA fragments encoding VH and VL segments can be further manipulated bystandard recombinant DNA techniques, for example to convert the variableregion genes to full-length antibody chain genes, to Fab fragment genesor to a scFv gene. In these manipulations, a VL- or VH-encoding DNAfragment is operably linked to another DNA fragment encoding anotherprotein, such as an antibody constant region or a flexible linker. Theterm “operably linked”, as used in this context, means that the two DNAfragments are joined such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operably linking the VH-encoding DNA toanother DNA molecule encoding heavy chain constant regions (CH1, CH2 andCH3 in the case of IgG1). The sequences of human heavy chain constantregion genes are known in the art (see e.g., Kabat, et al. (1991)(supra)) and DNA fragments encompassing these regions can be obtained bystandard PCR amplification.

Isolated DNA encoding the VL region can be converted to a full-lengthlight chain gene by operably linking the VL-encoding DNA to another DNAmolecule encoding the light chain constant region, CL. The sequences ofhuman light chain constant region genes are known in the art (see e.g.,Kabat, et al. (1991) (supra)) and DNA fragments encompassing theseregions can be obtained by standard PCR amplification. The light chainconstant region can be a kappa or lambda constant region, but mostpreferably is a kappa constant region.

In each case the VH or VL domains may be operably linked to theirrespective constant regions (CH or CL) where the constant regions aresite-specific constant regions and provide a site-specific antibody. Inselected embodiments the resulting site-specific antibody will comprisetwo unpaired cysteines in the CL domain.

For long-term, high-yield production of recombinant proteins stableexpression is preferred. Accordingly, cell lines that stably express theselected antibody may be engineered using standard art recognizedtechniques and form part of the invention. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter or enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker. Anyof the selection systems well known in the art may be used, includingthe glutamine synthetase gene expression system (the GS system) whichprovides an efficient approach for enhancing expression under selectedconditions. The GS system is discussed in whole or part in connectionwith EP 0 216 846, EP 0 256 055, EP 0 323 997 and EP 0 338 841 and U.S.Pat. Nos. 5,591,639 and 5,879,936. Another compatible expression systemfor the development of stable cell lines is the Freedom™ CHO-S Kit (LifeTechnologies).

Once an antibody of the invention has been produced by recombinantexpression or any other of the disclosed techniques, it may be purifiedor isolated by methods known in the art in that it is identified andseparated and/or recovered from its natural environment and separatedfrom contaminants that would interfere with diagnostic or therapeuticuses for the antibody or related ADC. Isolated antibodies includeantibodies in situ within recombinant cells.

These isolated preparations may be purified using various art-recognizedtechniques, such as, for example, ion exchange and size exclusionchromatography, dialysis, diafiltration, and affinity chromatography,particularly Protein A or Protein G affinity chromatography.

Antibody Conjugates

As discussed above the antibody of the invention is conjugated with twocalicheamicin toxins to form an “antibody drug conjugate” (ADC) or“antibody conjugate”. The term “conjugate” is used broadly and means thecovalent association of a calicheamicin with the antibody of the instantinvention. Herein the association is effected through cysteine residuesat position 214 of the light chain constant regions.

It will be appreciated that the ADCs of the instant invention may beused to selectively deliver predetermined calicheamicin warheads totumorigenic cells and/or cells expressing SEZ6. As set forth herein theterms “drug” or “warhead” may be used interchangeably and will mean acalicheamicin molecule. A “payload” comprises the calicheamicin incombination with a non-cleavable linker compound that provides a stablepharmaceutical complex until the ADC reaches the target. Formula II (setforth above) is an exemplary payload with a calicheamicin warhead.

In preferred embodiments the disclosed ADC will direct the bound payload(e.g., Formula II) to the target site in a relatively unreactive,non-toxic state before releasing and activating the calicheamicin toxin.This targeted release of the warhead is preferably achieved throughstable conjugation of the payloads and relatively homogeneouscomposition of the ADC preparations which minimize over-conjugated toxicADC species. Coupled with a particularly stable non-cleavable druglinker that is designed to release the warhead upon delivery to thetumor site, the conjugates of the instant invention can substantiallyreduce undesirable non-specific toxicity. This advantageously providesfor relatively high levels of the active cytotoxin at the tumor sitewhile minimizing exposure of non-targeted cells and tissue therebyproviding enhanced efficacy.

The conjugate of the instant invention may be generally represented byFormula III:

Ab-[L-D]n

wherein:

a) Ab comprises an anti-SEZ6 antibody having a heavy chain of SEQ IDNO:3 and a light chain of SEQ ID NO:4;

b) [L-D] comprises the linker drug of Formula II covalently attached toAb; and

c) n is 2.

In some preferred embodiments the instant invention comprises selectiveconjugation of the calicheamicin payload to free cysteines usingstabilization agents in combination with mild reducing agents asdescribed herein. Such reaction conditions tend to provide morehomogeneous preparations with less non-specific conjugation andcontaminants and correspondingly less toxicity.

Calicheamicin Warhead

As discussed herein the antibodies of the invention are conjugated to acalicheamicin toxin. That is, the disclosed SEZ6 ADC of the inventionmay comprise the formula Ab-[L-D]n (Formula III) or a pharmaceuticallyacceptable salt thereof wherein D is calicheamicin or analog thereof inany of the molecular structures provided herein. As known in the art thecalicheamicins are a class of enediyne antitumor antibiotics derivedfrom the bacterium Micromonospora echinospora, including calicheamicinγ₁ ^(I), calicheamicin β₁ ^(Br), calicheamicin γ₁ ^(Br), calicheamicinα₂ ^(I), calicheamicin α₃ ^(I), calicheamicin β₁ ^(i) and calicheamicinδ₁ ^(i) were isolated and characterized. The structures of each of theforegoing calicheamicin analogs are well known in the art (e.g., see Leeet al., Journal of Antibiotics, July 1989 which is incorporated hereinby reference in its entirety) and are compatible with the calicheamicindrug linker constructs and antibody drug conjugates disclosed herein.

In general, calicheamicin γ¹ contains two distinct structural regions,each playing a specific role in the compound's biological activity. Thelarger of the two consists of an extended sugar residue, comprising fourmonosaccharide units and one hexasubstituted benzene ring; these arejoined together through a highly unusual series of glycosidic,thioester, and hydroxylamine linkages. The second structural region, theaglycon (known as calicheamicinone), contains a compact, highlyfunctionalized bicyclic core, housing a strained enediyne unit within abridging 10-member ring. This aglycon subunit further comprises anallylic trisulfide which, as described below, functions as an activatorto generate the cytotoxic form of the molecule.

By way of example the structure for trisulfide calicheamicin γ₁ ^(I) isshown immediately below in Formula IV:

As used herein the term “calicheamicin” shall be held to mean any one ofcalicheamicin γ₁ ^(I), calicheamicin β₁ ^(Br), calicheamicin γ₁ ^(Br),calicheamicin α₂ ^(I), calicheamicin α₃ ^(I), calicheamicin β₁ ^(i) andcalicheamicin δ₁ ^(i) along with N-acetyl derivatives, sulfide analogsand analogs thereof. Accordingly, as used herein, the term“calicheamicin” will be understood to encompass any calicheamicin foundin nature as well as calicheamicin molecules with a disulfide moietyhaving a point of attachment to another molecule (e.g., an antibody drugconjugate) and analogs thereof. By way of example, as used herein,calicheamicin γ^(I) is to be understood to be construed as comprisingthe following molecules:

wherein R¹ is defined as below.

It will be appreciated that any of the aforementioned compounds arecompatible with the teachings herein and may be used to fabricate thedisclosed calicheamicin drug linker constructs and antibody drugconjugates. In certain embodiments, such as shown in Formula II, thecalicheamicin component of the disclosed antibody drug conjugates willcomprise N-acetyl Calicheamicin γ₁ ^(I) (N-Ac calicheamicin).

Calicheamicins target nucleic acids and cause strand scission therebykilling the target cell. More specifically, calicheamicins have beenfound to bind the minor groove of DNA, where they then undergo areaction analogous to Bergman cyclization to generate a diradicalspecies. In this regard the aryl tetrasaccharide subunit serves todeliver the drug to its target, tightly binding to the minor groove ofdouble helical DNA as demonstrated by Crothers et al. (1999). When anucleophile (e.g. glutathione) attacks the central sulfur atom of thetrisulfide group, it causes a significant change in structural geometryand imposes a great deal of strain on the 10-member enediyne ring. Thisstrain is completely relieved by the enediyne undergoing acycloaromatization reaction, generating a highly-reactive 1,4-benzenoiddiradical and leading, eventually, to DNA cleavage by attractinghydrogen atoms from the deoxyribose DNA backbone which results in strandscission. Note that in the calicheamicin disulfide analog constructs ofthe instant invention the nucleophile cleaves the protected disulfidebond to produce the desired diradical.

More particularly it is understood that D expressly comprises any memberof the class of calicheamicin as known in the art wherein the terminal—S—S—S—CH₃ moiety may be replaced with —S—Si

, wherein the symbol

represents the point of attachment to a linker.

Thus, in certain embodiments, D is of the Formula V,

wherein R¹ is hydrogen, halogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl, —CF₃,—CCl₃, —CBr₃, —CI₃, —CN, —C(O)R^(1E), —NR^(1A)R^(1C), —C(O)OR^(1A),—C(O)NR^(1B)R^(1C), —SR^(1D), —SO_(n1)R^(1B) or —SO_(v1)NR^(1B)R^(1C).In certain selected embodiments R¹ will comprise H. In other selectedembodiments R¹ will comprise —C(O)CH₃.

R^(1A), R^(1B), R^(1C), R^(1D) and R^(1E) are independently hydrogen,halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH, —NH₂, —COOH, —CONH₂, —N(O)₂,—SH, —S(O)₃H, —S(O)₄H, —S(O)₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHS(O)₂H, —NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCCl₃,—OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

In some embodiments, R^(1B) and R^(1C) substituents bonded to the samenitrogen atom may optionally be joined to form a substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl. The symbol n1 is independently an integer from 0 to 4, thesymbol v1 is independently 1 or 2 and the symbol

represents the point of attachment to a linker.

With regard to Formula V it will be appreciated that the illustratedcompound comprises a disulfide calicheamicin analog (e.g., an N-acetylcalicheamicin analog such as shown in Formula II) preferably bound to adisulfide protective group (at the point of attachment represented by

) that is covalently bound to the remainder of the linker. The disulfideprotective group improves stability of the disulfide bond in thebloodstream and allows for effective synthesis of the disclosedcalicheamicin-linker constructs. In certain embodiments thecalicheamicin disulfide group is preferably protected by a short chainsubstituted or unsubstituted bifunctional aliphatic or aryl group(“disulfide protective group”) that provides stability (e.g., plasmastability) until the ADC reaches the target cell. More specifically theconfiguration of the disulfide protective group provides a degree ofsteric hindrance for the disulfide bond thereby reducing itssusceptibility to cleavage via thiol-disulfide exchange reactions. Inthis position the disulfide protective group covalently links thecalicheamicin disulfide group with the remainder of the non-cleavablelinker.

Upon reaching the target (e.g., a cancer cell) the linker willpreferably be severed or degraded to release the calicheamicin attachedto part of the linker through the disulfide protective group. In certainembodiments once the linker has been initially cleaved beyond thedisulfide protective group (i.e. distal from the calicheamicin) theremainder of the linker attached to the calicheamicin will be degradedunder physiological conditions to the point where the disulfide bond issevered (preferably intracellularly) followed by rearrangement andformation of the active biradical calicheamicin species. It is this formof the calicheamicin warhead that binds to the minor groove of thecellular DNA and induces the desired cytotoxic effects (See Walker etal., Biochemistry 89: 4608-4612, 5/92 which is incorporated herein inits entirety by reference).

Linker Compounds

As indicated above payloads compatible with the instant inventioncomprise one or more warheads and a non-cleavable linker associating thewarheads with the antibody targeting agent. Compatible non-cleavablelinkers covalently bind with the reactive residue on the antibody(preferably a cysteine or lysine) and calicheamicin through thedisulfide moiety.

In particularly preferred embodiments the linker will comprise selectednon-cleavable linkers. In certain embodiments the ADCs will comprisecompatible non-cleavable linkers containing amide linked polyethyleneglycol or alkyl spacers that liberate the calicheamicin payload duringlysosomal degradation of the ADC within the target cell. A particularlycompatible non-cleavable linker used in Formula II is shown immediatelybelow in Formula VII wherein the wavy line indicates the point ofattachment to the disulfide group of the calicheamicin.

Synthesis of Formula II, including the linker component, is shown inExample 3 below with attendant conditions.

Conjugation

Various methods are known in the art for conjugating a therapeuticcompound to a cysteine residue and will be apparent to the skilledartisan. Under basic conditions the cysteine residues will bedeprotonated to generate a thiolate nucleophile which may be reactedwith soft electrophiles such as maleimides and iodoacetamides.Generally, reagents for such conjugations may react directly with acysteine thiol to form the conjugated protein or with a linker drug toform a linker drug intermediate. In the case of a linker, severalroutes, employing organic chemistry reactions, conditions, and reagentsare known to those skilled in the art, including: (1) reaction of acysteine group of the protein of the invention with a linker reagent, toform a protein-linker intermediate, via a covalent bond, followed byreaction with an activated compound; and (2) reaction of a nucleophilicgroup of a compound with a linker reagent, to form a drug linkerintermediate, via a covalent bond, followed by reaction with a cysteinegroup of a protein of the invention.

Prior to conjugation, antibodies may be made reactive for conjugationwith linker reagents by treatment with a reducing agent such asdithiothreitol (DTT) or (tris(2-carboxyethyl)phosphine (TCEP). In otherembodiments additional nucleophilic groups can be introduced intoantibodies through the reaction of lysines with reagents, including butnot limited to, 2-iminothiolane (Traut's reagent), SATA, SATP orSAT(PEG)4, resulting in conversion of an amine into a thiol.

Conjugation reagents commonly include maleimide, haloacetyl,iodoacetamide succinimidyl ester, isothiocyanate, sulfonyl chloride,2,6-dichlorotriazinyl, pentafluorophenyl ester, and phosphoramidite,although other functional groups can also be used. In certainembodiments methods include, for example, the use of maleimides,iodoacetimides or haloacetyl/alkyl halides, aziridine, acryloylderivatives to react with the thiol of a cysteine to produce a thioetherthat is reactive with a compound. Disulphide exchange of a free thiolwith an activated piridyldisulphide is also useful for producing aconjugate (e.g., use of 5-thio-2-nitrobenzoic (TNB) acid). Preferably, amaleimide is used.

As discussed above site-specific antibodies or engineered antibodiesallow for conjugate preparations that exhibit enhanced stability andsubstantial homogeneity due, at least in part, to the provision ofengineered free cysteine site(s) and/or the novel conjugation proceduresset forth herein. Unlike conventional conjugation methodology that fullyor partially reduces each of the intrachain or interchain antibodydisulfide bonds to provide conjugation sites (and is fully compatiblewith the instant invention), the present invention additionally providesfor the selective reduction of certain prepared free cysteine sites andattachment of the drug linker to the same.

In this regard it will be appreciated that the conjugation specificitypromoted by the engineered sites and the selective reduction allows fora high percentage of site directed conjugation at the desired positions.Significantly some of these conjugation sites, such as those present inthe terminal region of the light chain constant region, are typicallydifficult to conjugate effectively as they tend to cross-react withother free cysteines. However, through molecular engineering andselective reduction of the resulting free cysteines, efficientconjugation rates may be obtained which considerably reduces unwantedhigh-DAR contaminants and non-specific toxicity. More generally theengineered constructs and disclosed novel conjugation methods comprisingselective reduction provide ADC preparations having improvedpharmacokinetics and/or pharmacodynamics and, potentially, an improvedtherapeutic index.

In certain embodiments site-specific constructs present free cysteine(s)which, when reduced, comprise thiol groups that are nucleophilic andcapable of reacting to form covalent bonds with electrophilic groups onlinker moieties such as those disclosed above. As discussed aboveantibodies of the instant invention preferably have reducible unpairedinterchain cysteines, i.e. cysteines providing such nucleophilic groups.Thus, in certain embodiments the reaction of free sulfhydryl groups ofthe reduced free cysteines and the terminal maleimido or haloacetamidegroups of compatible drug linkers will provide the desired conjugation.In such cases free cysteines of the antibodies may be made reactive forconjugation with linker reagents by treatment with a reducing agent suchas dithiothreitol (DTT) or (tris (2-carboxyethyl) phosphine (TCEP). Eachfree cysteine will thus present, theoretically, a reactive thiolnucleophile. While such reagents are particularly compatible with theinstant invention it will be appreciated that conjugation ofsite-specific antibodies may be achieved using various reactions,conditions and reagents generally known to those skilled in the art.

In addition, it has been found that the free cysteines of engineeredantibodies may be selectively reduced to provide enhanced site-directedconjugation and a reduction in unwanted, potentially toxic contaminants.More specifically “stabilizing agents” such as arginine have been foundto modulate intra- and inter-molecular interactions in proteins and maybe used, in conjunction with selected reducing agents (preferablyrelatively mild), to selectively reduce the free cysteines and tofacilitate site-specific conjugation as set forth herein. As used hereinthe terms “selective reduction” or “selectively reducing” may be usedinterchangeably and shall mean the reduction of free cysteine(s) withoutsubstantially disrupting native disulfide bonds present in theengineered antibody. In selected embodiments this selective reductionmay be effected by the use of certain reducing agents or certainreducing agent concentrations. In other embodiments selective reductionof an engineered construct will comprise the use of stabilization agentsin combination with reducing agents (including mild reducing agents). Itwill be appreciated that the term “selective conjugation” shall mean theconjugation of an engineered antibody that has been selectively reducedin the presence of a cytotoxin as described herein. In this respect theuse of such stabilizing agents (e.g., arginine) in combination withselected reducing agents can markedly improve the efficiency ofsite-specific conjugation as determined by extent of conjugation on theheavy and light antibody chains and DAR distribution of the preparation.Compatible antibody constructs and selective conjugation techniques andreagents are extensively disclosed in WO2015/031698 which isincorporated herein specifically as to such methodology and constructs.

While not wishing to be bound by any particular theory, such stabilizingagents may act to modulate the electrostatic microenvironment and/ormodulate conformational changes at the desired conjugation site, therebyallowing relatively mild reducing agents (which do not materially reduceintact native disulfide bonds) to facilitate conjugation at the desiredfree cysteine site(s). Such agents (e.g., certain amino acids) are knownto form salt bridges (via hydrogen bonding and electrostaticinteractions) and can modulate protein-protein interactions in such away as to impart a stabilizing effect that may cause favorableconformational changes and/or reduce unfavorable protein-proteininteractions. Moreover, such agents may act to inhibit the formation ofundesired intramolecular (and intermolecular) cysteine-cysteine bondsafter reduction thus facilitating the desired conjugation reactionwherein the engineered site-specific cysteine is bound to the drug(preferably via a linker). Since selective reduction conditions do notprovide for the significant reduction of intact native disulfide bonds,the subsequent conjugation reaction is naturally driven to therelatively few reactive thiols on the free cysteines (e.g., preferably 2free thiols per antibody). As previously alluded to, such techniques maybe used to considerably reduce levels of non-specific conjugation andcorresponding unwanted DAR species in conjugate preparations fabricatedin accordance with the instant disclosure.

In selected embodiments stabilizing agents compatible with the presentinvention will generally comprise compounds with at least one moietyhaving a basic pKa. In certain embodiments the moiety will comprise aprimary amine while in other embodiments the amine moiety will comprisea secondary amine. In still other embodiments the amine moiety willcomprise a tertiary amine or a guanidinium group. In other selectedembodiments the amine moiety will comprise an amino acid while in othercompatible embodiments the amine moiety will comprise an amino acid sidechain. In yet other embodiments the amine moiety will comprise aproteinogenic amino acid. In still other embodiments the amine moietycomprises a non-proteinogenic amino acid. In some embodiments,compatible stabilizing agents may comprise arginine, lysine, proline andcysteine. In certain preferred embodiments the stabilizing agent willcomprise arginine. In addition, compatible stabilizing agents mayinclude guanidine and nitrogen containing heterocycles with basic pKa.

In certain embodiments compatible stabilizing agents comprise compoundswith at least one amine moiety having a pKa of greater than about 7.5,in other embodiments the subject amine moiety will have a pKa of greaterthan about 8.0, in yet other embodiments the amine moiety will have apKa greater than about 8.5 and in still other embodiments thestabilizing agent will comprise an amine moiety having a pKa of greaterthan about 9.0. Other embodiments will comprise stabilizing agents wherethe amine moiety will have a pKa of greater than about 9.5 while certainother embodiments will comprise stabilizing agents exhibiting at leastone amine moiety having a pKa of greater than about 10.0. In still otherembodiments the stabilizing agent will comprise a compound having theamine moiety with a pKa of greater than about 10.5, in other embodimentsthe stabilizing agent will comprise a compound having an amine moietywith a pKa greater than about 11.0, while in still other embodiments thestabilizing agent will comprise an amine moiety with a pKa greater thanabout 11.5. In yet other embodiments the stabilizing agent will comprisea compound having an amine moiety with a pKa greater than about 12.0,while in still other embodiments the stabilizing agent will comprise anamine moiety with a pKa greater than about 12.5. Those of skill in theart will understand that relevant pKa's may readily be calculated ordetermined using standard techniques and used to determine theapplicability of using a selected compound as a stabilizing agent.

The disclosed stabilizing agents are shown to be particularly effectiveat targeting conjugation to free site-specific cysteines when combinedwith certain reducing agents. For the purposes of the instant invention,compatible reducing agents may include any compound that produces areduced free site-specific cysteine for conjugation withoutsignificantly disrupting the native disulfide bonds of the engineeredantibody. Under such conditions, preferably provided by the combinationof selected stabilizing and reducing agents, the activated drug linkeris largely limited to binding to the desired free site-specific cysteinesite(s). Relatively mild reducing agents or reducing agents used atrelatively low concentrations to provide mild conditions areparticularly preferred. As used herein the terms “mild reducing agent”or “mild reducing conditions” shall be held to mean any agent or statebrought about by a reducing agent (optionally in the presence ofstabilizing agents) that provides thiols at the free cysteine site(s)without substantially disrupting native disulfide bonds present in theengineered antibody. That is, mild reducing agents or conditions(preferably in combination with a stabilizing agent) are able toeffectively reduce free cysteine(s) (provide a thiol) withoutsignificantly disrupting the protein's native disulfide bonds. Thedesired reducing conditions may be provided by a number ofsulfhydryl-based compounds that establish the appropriate environmentfor selective conjugation. In embodiments mild reducing agents maycomprise compounds having one or more free thiols while in someembodiments mild reducing agents will comprise compounds having a singlefree thiol. Non-limiting examples of reducing agents compatible with theselective reduction techniques of the instant invention compriseglutathione, n-acetyl cysteine, cysteine, 2-aminoethane-1-thiol and2-hydroxyethane-1-thiol.

It will further be appreciated that engineered antibodies capable ofconjugation may contain free cysteine residues that comprise sulfhydrylgroups that are blocked or capped as the antibody is produced or stored.Such caps include small molecules, proteins, peptides, ions and othermaterials that interact with the sulfhydryl group and prevent or inhibitconjugate formation. In some cases the unconjugated engineered antibodymay comprise free cysteines that bind other free cysteines on the sameor different antibodies. As discussed herein such cross-reactivity maylead to various contaminants during the fabrication procedure. In someembodiments, the engineered antibodies may require uncapping prior to aconjugation reaction. In specific embodiments, antibodies herein areuncapped and display a free sulfhydryl group capable of conjugation. Inspecific embodiments, antibodies herein are subjected to an uncappingreaction that does not disturb or rearrange the naturally occurringdisulfide bonds. It will be appreciated that in most cases the uncappingreactions will occur during the normal reduction reactions (reduction orselective reduction).

DAR Distribution and Purification

In selected embodiments conjugation and purification methodologycompatible with the present invention advantageously provides theability to generate relatively homogeneous ADC preparations comprising anarrow DAR distribution. In this regard the disclosed constructs (e.g.,site-specific constructs) and/or selective conjugation provides forhomogeneity of the ADC species within a sample in terms of thestoichiometric ratio between the drug and the engineered antibody andwith respect to the toxin location. As briefly discussed above the term“drug to antibody ratio” or “DAR” refers to the molar ratio of drug toantibody in an ADC preparation. In certain embodiments a conjugatepreparation may be substantially homogeneous with respect to its DARdistribution, meaning that within the ADC preparation is a predominantspecies of site-specific ADC with a particular drug loading (e.g., adrug loading of 2) that is also uniform with respect to the site ofloading (i.e., on the free cysteines). In other certain embodiments ofthe invention it is possible to achieve the desired homogeneity throughthe use of site-specific antibodies and/or selective reduction andconjugation. In other embodiments the desired homogeneity may beachieved through the use of site-specific constructs in combination withselective reduction. In yet other embodiments compatible preparationsmay be purified using analytical or preparative chromatographytechniques to provide the desired homogeneity. In each of theseembodiments the homogeneity of the ADC sample can be analyzed usingvarious techniques known in the art including but not limited to massspectrometry, HPLC (e.g. size exclusion HPLC, RP-HPLC, HIC-HPLC etc.) orcapillary electrophoresis.

With regard to the purification of ADC preparations it will beappreciated that standard pharmaceutical preparative methods may beemployed to obtain the desired purity. As discussed herein liquidchromatography methods such as reverse phase (RP) and hydrophobicinteraction chromatography (HIC) may separate compounds in the mixtureby drug loading value. In some cases, ion-exchange (IEC) or mixed-modechromatography (MMC) may also be used to isolate species with a specificdrug load.

In any event the disclosed ADCs and preparations thereof may comprisedrug and antibody moieties in various stoichiometric molar ratiosdepending on the configuration of the antibody and, at least in part, onthe method used to effect conjugation. In certain preferred embodimentsthe drug loading per ADC may comprise 2 calicheamicin warheads.

Despite the relatively high level of homogeneity provided by the instantinvention the disclosed compositions actually comprise a mixture ofconjugates with a range of drug compounds. As such, the disclosed ADCcompositions include mixtures of conjugates where most of theconstituent antibodies are covalently linked to one or more drugmoieties and (despite the relative conjugate specificity provided byengineered constructs and selective reduction) where the drug moietiesmay be attached to the antibody by various thiol groups. That is,following conjugation, compositions of the invention will comprise amixture of ADCs with different drug loads at various concentrations(along with certain reaction contaminants primarily caused by freecysteine cross reactivity). However, using selective reduction andpost-fabrication purification the conjugate compositions may be drivento the point where they largely contain a single predominant desired ADCspecies (e.g., with a drug loading of 2) with relatively low levels ofother ADC species (e.g., with a drug loading of 1, 4, 6, etc.). Theaverage DAR value represents the weighted average of drug loading forthe composition as a whole (i.e., all the ADC species taken together).Those of skill in the art will appreciate that acceptable DAR values orspecifications are often presented as an average, a range ordistribution (i.e., an average DAR of 2+/−0.5). Preferably compositionscomprising a measured average DAR within the range (i.e., 1.5 to 2.5)would be used in a pharmaceutical setting.

Thus, in some embodiments the present invention will comprisecompositions having an average DAR of 2+/−0.5. In other embodiments thepresent invention will comprise an average DAR of 2+/−0.4 or a DAR of2+/−0.3 or a DAR of 2+/−0.2. In other embodiments IgG1 conjugatecompositions will preferably comprise a composition with relatively lowlevels (i.e., less than 30%) of non-predominant ADC species (e.g., ADCswith a drug loading of 0, 1, 3, 4, 5, etc.). In some embodiments the ADCcomposition will comprise an average DAR of 2+/−0.4 with relatively lowlevels (<30%) of non-predominant ADC species. In some embodiments theADC composition will comprise an average DAR of 2+/−0.3 with relativelylow levels (<30%) of non-predominant ADC species. In yet otherembodiments the predominant ADC species (e.g., with a drug loading of 2)will be present at a concentration of greater than 50%, at aconcentration of greater than 55%, at a concentration of greater than60%, at a concentration of greater than 65%, at a concentration ofgreater than 70%, at a concentration of greater than 75%, at aconcentration of greater that 80%, at a concentration of greater than85%, at a concentration of greater than 90%, at a concentration ofgreater than 93%, at a concentration of greater than 95% or even at aconcentration of greater than 97% when measured against all other DARspecies present in the composition.

As detailed in the Examples below the distribution of drugs per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as UV-Vis spectrophotometry, reverse phaseHPLC, HIC, mass spectroscopy, ELISA, and electrophoresis. Thequantitative distribution of ADC in terms of drugs per antibody may alsobe determined.

Pharmaceutical Preparations and Therapeutic Uses

The antibodies or ADCs of the invention can be formulated in variousways using art recognized techniques. In some embodiments, thetherapeutic compositions of the invention can be administered neat orwith a minimum of additional components while others may optionally beformulated to contain suitable pharmaceutically acceptable carriers. Asused herein, “pharmaceutically acceptable carriers” comprise excipients,vehicles, adjuvants and diluents that are well known in the art.

Dosages and Dosing Regimens

The particular dosage regimen, i.e., dose, timing and repetition, willdepend on the particular individual, as well as empirical considerationssuch as pharmacokinetics (e.g., half-life, clearance rate, etc.).Determination of the frequency of administration may be made by personsskilled in the art, such as an attending physician based onconsiderations of the condition and severity of the condition beingtreated, age and general state of health of the subject being treatedand the like. Frequency of administration may be adjusted over thecourse of therapy based on assessment of the efficacy of the selectedcomposition and the dosing regimen. Such assessment can be made on thebasis of markers of the specific disease, disorder or condition. Inembodiments where the individual has cancer, these include directmeasurements of tumor size via palpation or visual observation; indirectmeasurement of tumor size by x-ray or other imaging techniques; animprovement as assessed by direct tumor biopsy and microscopicexamination of a tumor sample; the measurement of an indirect tumormarker or an antigen identified according to art-recognized techniques;reduction in the number of proliferative or tumorigenic cells,maintenance of the reduction of such neoplastic cells; reduction of theproliferation of neoplastic cells; or delay in the development ofmetastasis.

Indications

The invention provides for the use of an ADC of the invention for thetreatment of various neoplastic disorders. In certain embodiments thediseases to be treated are neoplastic conditions comprising solidtumors. In selected embodiments the ADC of the invention will be used totreat tumors or tumorigenic cells expressing a SEZ6 determinant. Incertain other embodiments the disclosed ADC will be used to treat asubject suffering from small cell lung cancer (SCLC). Preferably the“subject” or “patient” to be treated will be human although, as usedherein, the terms are expressly held to comprise any mammalian species.

In selected embodiments the ADC can be administered to small cell lungcancer patients exhibiting limited stage disease or extensive stagedisease. In other embodiments the disclosed ADC will be administered torefractory patients (i.e., those whose disease recurs during or shortlyafter completing a course of initial therapy); sensitive patients (i.e.,those whose relapse is longer than 2-3 months after primary therapy); orpatients exhibiting resistance to a platinum based agent (e.g.carboplatin, cisplatin, oxaliplatin) and/or a taxane (e.g. docetaxel,paclitaxel, larotaxel or cabazitaxel). In certain preferred embodimentsthe SEZ6 ADC of the instant invention may be administered to frontlinepatients. In other embodiments the SEZ6 ADC of the instant invention maybe administered to second line patients. In still other embodiments theSEZ6 ADC of the instant invention may be administered to third linepatients or to fourth line patients.

Articles of Manufacture

The invention includes pharmaceutical packs and kits comprising one ormore containers or receptacles, wherein a container can comprise one ormore doses of the ADC of the invention. In certain embodiments, the packor kit contains a unit dosage, meaning a predetermined amount of acomposition comprising, for example, the ADC of the invention, with orwithout one or more additional agents and optionally, one or moreanti-cancer agents.

When the components of the kit are provided in one or more liquidsolutions, aqueous or non-aqueous though typically an aqueous solutionis preferred, with a sterile aqueous solution being particularlypreferred. The formulation in the kit can also be provided as driedpowder(s) or in lyophilized form that can be reconstituted upon additionof an appropriate liquid. The liquid used for reconstitution can becontained in a separate container. Such liquids can comprise sterile,pharmaceutically acceptable buffer(s) or other diluent(s) such asbacteriostatic water for injection. Where the kit comprises the ADC ofthe invention in combination with additional therapeutics or agents, thesolution may be pre-mixed, either in a molar equivalent combination, orwith one component in excess of the other. Alternatively, the ADC of theinvention and any optional anti-cancer agent or other agent can bemaintained separately within distinct containers prior to administrationto a patient.

In certain preferred embodiments the aforementioned kits, incorporatingcompositions of the invention will comprise a label, marker, packageinsert, bar code and/or reader indicating that the kit contents may beused for the treatment of cancer. In other preferred embodiments the kitmay comprise a label, marker, package insert, bar code and/or readerindicating that the kit contents may be administered in accordance witha certain dosage or dosing regimen to treat a subject suffering fromcancer. In other particularly preferred aspects the label, marker,package insert, bar code and/or reader indicates that the kit contentsmay be used for the treatment of small cell lung cancer or a dosingregimen for treatment of the same.

Suitable containers or receptacles include, for example, bottles, vials,syringes, infusion bags (i.v. bags), etc. The containers can be formedfrom a variety of materials such as glass or pharmaceutically compatibleplastics. In certain embodiments the receptacle(s) can comprise asterile access port. For example, the container may be an intravenoussolution bag or a vial having a stopper that can be pierced by ahypodermic injection needle.

In some embodiments the kit can contain a means by which to administerthe ADC and any optional components to a patient, e.g., one or moreneedles or syringes (pre-filled or empty), or other such like apparatus,from which the formulation may be injected or introduced into thesubject or applied to a diseased area of the body. The kits of theinvention will also typically include a means for containing the vials,or such like, and other components in close confinement for commercialsale, such as, e.g., blow-molded plastic containers into which thedesired vials and other apparatus are placed and retained.

Miscellaneous

Unless otherwise defined herein, scientific and technical terms used inconnection with the invention shall have the meanings that are commonlyunderstood by those of ordinary skill in the art. Further, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. In addition, rangesprovided in the specification and appended claims include both endpoints and all points between the end points. Therefore, a range of 2.0to 3.0 includes 2.0, 3.0, and all points between 2.0 and 3.0.

Generally, techniques of cell and tissue culture, molecular biology,immunology, microbiology, genetics and chemistry described herein arethose well-known and commonly used in the art. The nomenclature usedherein, in association with such techniques, is also commonly used inthe art. The methods and techniques of the invention are generallyperformed according to conventional methods well known in the art and asdescribed in various references that are cited throughout the presentspecification unless otherwise indicated.

REFERENCES

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forexample, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference, regardless of whether thephrase “incorporated by reference” is or is not used in relation to theparticular reference. The foregoing detailed description and theexamples that follow have been given for clarity of understanding only.No unnecessary limitations are to be understood therefrom. The inventionis not limited to the exact details shown and described. Variationsobvious to one skilled in the art are included in the invention definedby the claims. Any section headings used herein are for organizationalpurposes only and are not to be construed as limiting the subject matterdescribed.

8. EXAMPLES

The invention, generally described above, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the instantinvention. The examples are not intended to represent that theexperiments below are all or the only experiments performed. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Sequence Listing Summary

TABLE 2 provides a summary of amino acid and nucleic acid sequencesincluded herein.

TABLE 2 SEQ ID NO Description 1 Seizure protein 6 homolog isoform 1precursor (NP_849191) 2 Seizure protein 6 homolog isoform 2 precursor(NP_001092105) 3 hSEZ6-1.ss1 heavy chain protein sequence 4 hSEZ6-1.ss1light chain protein sequence 5 Nucleic acid sequence encodinghSEZ6-1.ss1 heavy chain protein sequence including introns 6 Nucleicacid sequence encoding hSEZ6-1.ss1 light chain protein sequence

Example 1: Generation of a SEZ6 Antibody

SEZ6 murine antibodies were produced in accordance with the teachingsherein through inoculation with human SEZ6-Fc. In this regard threestrains of mice were used to generate high affinity, murine, monoclonalantibody modulators that can be used to associate with and/or inhibitthe action of human SEZ6 (e.g., NP_849191: Seizure protein 6 homologisoform 1 precursor; NP 001092105: Seizure protein 6 homolog isoform 2precursor) for the prevention and/or treatment of various proliferativedisorders. Specifically, Balb/c, CD-1 and FVB mouse strains wereimmunized with human recombinant SEZ6-Fc and used to produce Hybridomas.

The SEZ6-Fc antigen was purified from supernatant from CHO-S cells overexpressing a SEZ6-Fc construct. 10 μg of SEZ6-Fc immunogen was used forthe first immunization, followed by 5 μg and 2.5 μg of SEZ6-Fc immunogenfor the subsequent three immunizations and five immunizations,respectively. All immunizations were performed with the immunogenemulsified with an equal volume of TITERMAX® Gold (CytRx Corporation) oralum adjuvant. Murine antibodies were generated by immunizing six femalemice (two each of: Balb/c, CD-1, FVB) via footpad route for allinjections.

Solid-phase ELISA assays were used to screen mouse sera for mouse IgGantibodies specific for human SEZ6. A positive signal above backgroundwas indicative of antibodies specific for SEZ6. Briefly, 96 well plates(VWR International, Cat. #610744) were coated with recombinant SEZ6-Hisat 0.5 μg/ml in ELISA coating buffer overnight. After washing with PBScontaining 0.02% (v/v) Tween 20, the wells were blocked with 3% (w/v)BSA in PBS, 200 μL/well for 1 hour at room temperature (RT). Mouse serumwas titrated (1:100, 1:200, 1:400, and 1:800) and added to the SEZ6coated plates at 50 μL/well and incubated at RT for 1 hour. The platesare washed and then incubated with 50 μL/well HRP-labeled goatanti-mouse IgG diluted 1:10,000 in 3% BSA-PBS or 2% FCS in PBS for 1hour at RT. Again the plates were washed and 40 μL/well of a TMBsubstrate solution (Thermo Scientific 34028) was added for 15 minutes atRT. After developing, an equal volume of 2N H₂SO₄ was added to stopsubstrate development and the plates were analyzed by spectrophotometerat OD 450.

Sera-positive immunized mice were sacrificed and draining lymph nodes(popliteal and inguinal, and medial iliac if enlarged) were dissectedout and used as a source for antibody producing cells. A single cellsuspension of B cells (228.9×10⁶ cells) was fused with non-secretingP3x63Ag8.653 myeloma cells (ATCC #CRL-1580) at a ratio of 1:1 byelectrofusion. Electrofusion was performed using the BTX Hybrimmune™System, (BTX Harvard Apparatus) as per the manufacturer's directions.After the fusion procedure the cells were resuspended in hybridomaselection medium supplemented with Azaserine (Sigma #A9666), highglucose DMEM medium with sodium pyruvate (Cellgro cat #15-017-CM)containing 15% Fetal Clone I serum (Hyclone), 10% BM Condimed (RocheApplied Sciences), 4 mM L-glutamine, 100 IU Penicillin-Streptomycin and50 μM 2-mercaptoethanol and then plated in three T225 flasks in 90 mLselection medium per flask. The flasks were then placed in a humidified37° C. incubator containing 5% CO₂ and 95% air for 6-7 days.

After six to seven days of growth the library consisting of the cellsgrown in bulk in the T225s was plated at 1 cell per well in Falcon 96well U-bottom plates using the Aria I cell sorter. The selectedhybridomas were then grown in 200 μL of culture medium containing 15%Fetal Clone I serum (Hyclone), 10% BM-Condimed (Roche Applied Sciences),1 mM sodium pyruvate, 4 mM L-glutamine, 100 IU Penicillin-Streptomycin,50 μM 2-mercaptoethanol, and 100 μM hypoxanthine Any remaining unusedhybridoma library cells were frozen for future library testing. Afterten to eleven days of growth supernatants from each well of the platedcells were assayed for antibodies reactive for SEZ6 by ELISA and FACSassays.

For screening by ELISA 96 well plates were coated with SEZ6-Fc at 0.3μg/mL in PBS overnight at 4° C. The plates were washed and blocked with3% BSA in PBS/Tween for one hour at 37° C. and used immediately or keptat 4° C. Undiluted hybridoma supernatants were incubated on the platesfor one hour at RT. The plates were washed and probed with HRP labeledgoat anti-mouse IgG diluted 1:10,000 in 3% BSA-PBS for one hour at RT.The plates were then incubated with substrate solution as describedabove and read at OD 450. Wells containing immunoglobulin thatpreferentially bound human SEZ6, as determined by a signal abovebackground, were transferred and expanded.

Selected growth positive hybridoma wells secreting murine immunoglobulinwere also screened for human SEZ6 specificity and cynomolgus, rat andmurine SEZ6 cross reactivity using a flow cytometry based assay with 293cells engineered to over-express the selected species specific antigen.

For the flow cytometry assays, 50×10⁴ h293 cells transduced respectivelywith human, cynomolgus, rat or murine SEZ6 were incubated for 30 minuteswith 25-100 μL hybridoma supernatant. Cells were washed with PBS, 2%FCS, twice and then incubated with 50 μL of a goat-anti-mouse IgG Fcfragment specific secondary conjugated to DyLight 649 diluted 1:200 inPBS/2% FCS. After 15 minutes of incubation, cells were washed twice withPBS, 2% FCS, and re-suspended in the same buffer with DAPI and analyzedby flow cytometry using a FACSCanto II as per the manufacturer'sinstructions. Wells containing immunoglobulin that preferentially boundthe SEZ6⁺ GFP⁺ cells were transferred and expanded. The resulting hSEZ6specific clonal hybridomas were cryopreserved in CS-10 freezing medium(Biolife Solutions) and stored in liquid nitrogen. Antibodies that boundwith human, cynomolgus, rat or murine SEZ6 cells were noted ascross-reactive.

ELISA and flow cytometry analysis confirmed that purified antibody frommost or all of these hybridomas bound SEZ6 in a concentration-dependentmanner. Wells containing immunoglobulin that bound SEZ6 GFP cells weretransferred and expanded. The resulting clonal hybridomas werecryopreserved in CS-10 freezing medium (Biolife Solutions) and stored inliquid nitrogen.

One fusion was performed and seeded in 48 plates (4608 wells atapproximately 40% cloning efficiency). The initial screen yieldedsixty-three murine antibodies that associated with human SEZ6. A secondscreen was subsequently performed and yielded 134 antibodies thatassociated with human SEZ6.

Example 2: Fabrication of a Humanized Site-Specific SEZ6 Antibody

An antibody from Example 1 was chosen for further processing andhumanization. RNA from the hybridoma expressing the selected antibodywas isolated, amplified and sequenced using standard art-recognizedtechniques. From the nucleotide sequence information, data regarding V,D and J gene segments of the heavy and light chains of subject murineantibodies were obtained. The V-(D)-J sequences were aligned with mouseIg germ line sequences and acceptor human variable framework regionswere selected based on their highest sequence homology to the subjectmouse framework sequence and its canonical structure for CDR grafting.The resulting genetic arrangement for the humanized variable regions ofthe antibody are shown in Table 3A immediately below.

TABLE 3A human human FW human human FW mAb VH JH changes VK JK changesSEZ6-1 IGHV5-51 JH4 none IGKV-L6 JK4 none

The engineered variable regions were then used to generate a humanIgG1/kappa anti-SEZ6 site-specific antibody comprising a native kappalight chain (LC) constant region and a heavy chain (HC) constant regionmutated to provide an unpaired cysteine. In this regard cysteine 220(C220) in the upper hinge region of the HC was substituted with serine(C220S) to provide the hSEZ6-1.ss1 antibody. When assembled, the HC andLC form an antibody comprising two free cysteines at the c-terminal endsof the light chain constant regions (e.g., C214) that are suitable forconjugation to a therapeutic agent. Unless otherwise noted all numberingof constant region residues is in accordance with the EU numberingscheme as set forth in Kabat et al.

To generate the site-specific constructs a VH nucleic acid was clonedonto an expression vector containing a C220S mutated HC constant region.Resulting vectors encoding the mutant C220S HC were co-transfected inCHO-S cells with a vector encoding the selected light chain variableregion operably associated with a wild-type IgG1 kappa LC and expressedusing a mammalian transient expression system.

In addition to the C220S mutation to provide the free cysteines in theconstant region, two additional modifications were made on the heavychain. First, the C-terminal lysine was deleted in order to reduceheterogeneity in expression. Second, a conservative mutation was made inthe heavy chain variable region to improve molecular stability andfacilitate antibody production. More specifically, a conservativemutation was incorporated in the heavy chain CDR2 (as defined by Kabat)to eliminate a canonical glycosylation site. Glycosylation at this sitecould potentially impart heterogeneity in the expressed protein whichmay result in a reduction in binding affinity. Accordingly, asubstitution of serine to asparagine at Kabat position 60 (S60N) wasincorporated into the heavy chain to eliminate the glycosylation site.The resulting humanized antibody with these mutations was termedhSEZ6-1.ss1. As discussed in more detail below, substantial equivalencyof hSEZ6-1.ss1 to the parental humanized antibody and murine sourceantibody was confirmed as to affinity.

The resulting genetic arrangement for the humanized variable regions ofthe mutated antibody are shown in Table 3B immediately below.

TABLE 3B human human FW CDR Changes human human FW CDR Changes mAb VH JHchanges (VH) VK JK changes (VK) SEZ6-1.ss1 IGHV5-51 JH4 none S60NIGKV-L6 JK4 none none

The amino acid sequence of the full-length hSEZ6-1.ss1 site-specificantibody heavy chain is SEQ ID NO:3, having the variable region mutationsite S60N and constant region mutation site C220S. The amino acidsequence of the heavy chain variable region is shown below as SEQ IDNO:7, having the S60N mutation underlined.

(SEQ ID NO: 7) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSSWINWVRQMPGKGLEWMGRIYPGEGDTNY N GNFEGQVTISADKSISTAYLQWSSLKASDTAMYYCTR GLVMDYWGQGTLVTVSSThe amino acid sequence of the full-length hSEZ6-1.ss1 site-specificantibody light chain is SEQ ID NO:4, having the toxin conjugationresidue at C214. The amino acid sequence of the light chain variableregion is shown below as SEQ ID NO:8

(SEQ ID NO: 8) EIVLTQSPATLSLSPGERATLSCRASQSVDYNGISYMHVVYQQKPGQAPRLLIYAASNVQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSIE DPPTFGGGTKVEIK

Example 3: Preparation of hSEZ6-1.ss1 Drug Linker

A drug linker compound according to Formula II

was synthesized as set forth immediately below.

Step 1. tert-butyl[34-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,32-dioxo-7,10,13,16,19,22,25,28-octaoxa-3,31-diazatetratriacontan-1-yl]carbamate

3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-{27-[(2,5-dioxopyrrolidin-1-yl)oxy]-27-oxo-3,6,9,12,15,18,21,24-octaoxaheptacosan-1-yl}propanamide(434 mg) was dissolved in N,N-dimethylformamide (5 mL) and treated withtert-butyl (2-aminoethyl)carbamate (108.1 mg). After 6 hours thereaction mixture was concentrated, and the residue purified by silicagel chromatography eluted with 0% CH3OH/CH₂Cl₂ to 10% CH3OH/CH₂Cl₂ togive the titled compound (154.2 mg). LC/MS (Analytical method A):Rt=1.65 min, m/z 735.46 [M+H]⁺.

Step 2.N-(2-aminoethyl)-31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azahentriacontan-1-amide

The tert-butoxycarbonyl protected amine (Step 1, 154.2 mg) was dissolvedin N,N-dimethylformamide (5 mL), trifluoroacetic acid (500 μL) was addedover 30 seconds, and the resultant mixture was stirred for 30 minutes.After reaction completion, the reaction mixture was concentrated andused without further purification. LC/MS (Analytical method A): Rt=1.29min, m/z 635.39 [M+H]⁺.

Step 3.4-{[(2E)-2-{(1R,4Z,8S)-8-({(2R,3R,4S,5S,6R)-3-({(2S,4S,5S)-5-[acetyl(ethyl)amino]-4-methoxyoxan-2-yl}oxy)-5-[({(2S,4S,5S,6R)-5-[(4-{[(2S,3R,4R,5S,6S)-3,5-dihydroxy-4-methoxy-6-methyloxan-2-yl]oxy}-3-iodo-5,6-dimethoxy-2-methylbenzoyl)sulfanyl]-4-hydroxy-6-methyloxan-2-yl}oxy)amino]-4-hydroxy-6-methyloxan-2-yl}oxy)-1-hydroxy-10-[(methoxycarbonyl)amino]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-ylidene}ethyl]disulfanyl}-4-methylpentanoicacid

N-Acetyl calicheamicin γ 1 (0.2 g, 0.142 mmol, 1 eq) was dissolved inacetonitrile (30 mL), and the resultant solution was chilled to −15° C.4-Mercapto-4-methylpentanoic acid (0.420 mL, 2.837 mmol, 20 eq) wasdissolved in acetonitrile (10 mL) and added slowly to the cooledsolution of N-acetyl calicheamicin γ 1. Triethylamine (0.377 mL, 2.837mmol, 20 eq) was added to the reaction mixture, and then the reactionmixture was allowed to warm up to room temperature over 3-18 hours. Uponcompletion of the reaction, the mixture was concentrated, and theresidue was dry loaded onto silica gel for flash chromatographypurification eluted with 2-20% methanol/dichloromethane to give thetitled compound. The titled compound was precipitated out of colddiethyl ether. LC/MS (analytical method A): Rt=1.92 min, m/z 1478.64[M+H]⁺.

Step 4.S-[(2R,3S,4S,6S)-6-({[(2R,3S,4S,5R,6R)-5-({(2S,4S,5S)-5-[acetyl(ethyl)amino]-4-methoxyoxan-2-yl}oxy)-6-{[(2S,5Z,9R,13E)-13-[43-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,5-dimethyl-8,13,41-trioxo-16,19,22,25,28,31,34,37-octaoxa-3,4-dithia-9,12,40-triazatritetracontan-1-ylidene]-9-hydroxy-12-[(methoxycarbonyl)amino]-11-oxobicyclo[7.3.1]trideca-1(12),5-diene-3,7-diyn-2-yl]oxy}-4-hydroxy-2-methyloxan-3-yl]amino}oxy)-4-hydroxy-2-methyloxan-3-yl]4-{[(2S,3R,4R,5S,6S)-3,5-dihydroxy-4-methoxy-6-methyloxan-2-yl]oxy}-3-iodo-5,6-dimethoxy-2-methylbenzene-1-carbothioate

N-Acetyl calicheamicin acid (Step 3, 100 mg, 0.068 mmol, 1 eq) wasdissolved in N,N-dimethylformamide (3.4 mL) and cooled to 0° C.N,N-Diisopropylethylamine (176 μL, 1.01 mmol, 15 eq) and(1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbeniumhexafluorophosphate (COMU, 43 mg, 0.1 mmol, 1.5 eq) were thensequentially added. After 2 minutes, the N-(2-aminoethyl)-31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azahentriacontan-1-amide(Step 2, 51.4 mg, 0.08 mmol, 1.2 eq) in N,N-dimethylformamide (200 μL)was added. After 1 hour, the reaction mixture was concentrated, and theresidue was purified by preparative HPLC (method pA) to give the titledcompound (16.8 mg, 12% yield). LC/MS (Analytical method B or C): Rt=8.18min. HRMS calculated [M+H]+=2094.7049, Observed [M+H]⁺=2094.6902. ¹H NMR(400 MHz, DMSO-d₆) δ 9.03 (s, 1H), 8.01 (t, J=5.5 Hz, 1H), 7.86 (s, 2H),7.01 (s, 3H), 6.80 (d, J=8.0 Hz, 1H), 6.30-6.18 (m, 1H), 6.13 (dd,J=9.5, 7.1 Hz, 1H), 6.09-5.99 (m, 2H), 5.56 (d, J=4.0 Hz, 1H), 5.45 (s,1H), 5.43-5.37 (m, 2H), 5.12 (dd, J=13.4, 5.1 Hz, 2H), 4.94 (d, J=9.9Hz, 1H), 4.63-4.47 (m, 2H), 4.27-4.13 (m, 2H), 4.11 (s, 1H), 4.08-3.97(m, 1H), 3.91 (dd, J=10.8, 6.1 Hz, 1H), 3.81 (s, 3H), 3.78-3.81 (m, 1H),3.77 (s, 3H), 3.73-3.63 (m, 1H), 3.63-3.55 (m, 7H), 3.55-3.46 (m, 30H),3.41 (s, 3H), 3.25 (d, J=2.3 Hz, 3H), 3.15 (q, J=5.8 Hz, 2H), 3.07 (s,5H), 2.93 (d, J=17.7 Hz, 1H), 2.47-2.38 (m, 1H), 2.36-2.26 (m, 7H),2.15-2.06 (m, 2H), 2.01 (d, J=2.7 Hz, 3H), 1.87 (d, J=12.3 Hz, 1H),1.80-1.62 (m, 3H), 1.26 (dd, J=6.1, 3.3 Hz, 4H), 1.24-1.19 (m, 2H),1.19-1.12 (m, 7H), 1.09 (t, J=7.1 Hz, 2H), 0.95 (t, J=6.9 Hz, 1H).

General Information on analytical and preparative HPLC methods.

Analytical Method A:

MS: Waters® Acuity® Ultra SQ Detector ESI, Scan range 120-2040 Da.

Column: Waters Acuity UPLC® BEH C18, 1.7 μm, 2.1×50 mm

Column temperature: 50° C.

Flow rate: 0.6 mL/min

Mobile phase A: 0.1% formic acid in water.

Mobile phase B: 0.1% formic acid in acetonitrile.

Gradient:

Time, minutes % A % B 0 95 5 0.25 95 5 2 0 100 2.5 0 100 3 95 5 4 95 5

Analytical Method B:

MS: Waters® Acuity® Ultra SQ Detector ESI, Scan range 120-2040 Da,

Column: Waters Acuity UPLC® BEH C18, 1.7 μm, 2.1×50 mm

Column temperature: 60° C.

Flow rate: 0.4 mL/min

Mobile phase A: 0.1% formic acid in water.

Mobile phase B: 0.1% formic acid in acetonitrile.

Gradient:

Time, minutes % A % B 0 95 5 2 95 5 3 80 20 13 20 80 14 20 80 14.10 5 9515 5 95 15.10 95 5 20 95 5

Analytical Method C:

HRMS: AB Sciex 5600 Plus Triple Time-of-Flight (TOF), scan range250-2500 Da

Column: Waters Acuity UPLC® BEH C18, 1.7 μm, 2.1×50 mm

Column temperature: 60° C.

Flow rate: 0.4 mL/min

Mobile phase A: 0.1% formic acid in water.

Mobile phase B: 0.1% formic acid in acetonitrile.

Gradient:

Time, minutes % A % B 0 95 5 2 95 5 3 80 20 13 20 80 14 20 80 14.10 5 9515 5 95 15.10 95 5 20 95 5

Analytical Method D

Column: EMD Millipore Chromolith® Flash RP-18 endcapped 25-2 mm.

Column temperature: 40° C.

Wavelength: 220 nm

Flow rate: 1.5 mL/minute

Mobile phase A: H₂O (4 L with 1.5 mL trifluoroacetic acid)

Mobile phase B: acetonitrile (4 L with 0.75 mL trifluoroacetic acid)

Gradient:

Time, minutes % A % B 0 95 5 0.01 95 5 0.70 5 95 1.15 5 95 1.16 95 51.60 95 5

Analytical Method E

Column: Halo C18 2.1×30 mm, 2.7 μm

Detection: diode array and positive/negative electrospray ionization

Flow rate: 1.0 mL/minute

Mobile phase A: 0.0375% trifluoroacetic acid in water

Mobile phase B: 0.018% trifluoroacetic acid in acetonitrile

Gradient:

Time, minutes % A % B 0 90 10 2.00 20 80 2.48 20 80 2.50 90 10 3.00 9010

Analytical Method F

Column: Venusil XBP-C18, 2.1×50 mm, 5 μm

Detection: diode array and positive/negative electrospray ionization

Flow rate: 0.8 mL/minute

Mobile phase A: 0.0375% trifluoroacetic acid in water

Mobile phase B: 0.018% trifluoroacetic acid in acetonitrile

Gradient:

Time, minutes % A % B 0 99 1 3.40 10 90 3.85 0 100 3.86 99 1 4.51 99 1

Preparative HPLC Method pA:

Column: Waters XBridge™ prep C18 5 μm OBD, 19×100 mm

Column temperature: ambient

Flow rate: 15 mL/min

Mobile phase A: 0.1% formic acid in water.

Mobile phase B: 0.1% formic acid in acetonitrile.

Gradient:

Time, min % A % B 0 95 5 5 95 5 8 80 20 50 20 80 52.59 20 80 52.92 5 9555.87 5 95 56.20 95 5 60 95 5

Preparative HPLC Method pB:

Column: Phenomenex Luna® C18(2), 250×50 mm i.d., 10 μm

Wavelengths: 220 and 254 nm

Flow rate: 80 mL/minute

Mobile phase A: 0.01 M NH₄HCO₃ in H₂O.

Mobile phase B: acetonitrile

Gradient: 30-50% mobile phase B over 20 minutes

Preparative HPLC Method pC

Column: Phenomenex Luna® C18(2), 250×50 mm i.d., 10 μm

Wavelengths: 220 and 254 nm

Flow rate: 80 mL/minute

Mobile phase A: 0.075% v/v trifluoroacetic acid in water

Mobile phase B: acetonitrile

Gradient: 10-40% mobile phase B over 20 minutes

Preparative HLC Method pD

Column: Phenomenex Luna® C18(2), 250×50 mm i.d., 10 μm

Wavelengths: 220 and 254 nm

Flow rate: 80 mL/minute

Mobile phase A: 0.09% v/v trifluoroacetic acid in water

Mobile phase B: acetonitrile

Gradient: 15-43% mobile phase B over 20 minutes

Example 4: Preparation of hSEZ6-1.ss1 Antibody Drug Conjugates

Anti-hSEZ6-1.ss1 ADCs were prepared according to the teachings hereinfor further in vitro and in vivo testing.

In this regard hSEZ6-1.ss1 from Example 2 was conjugated to anon-cleavable calicheamicin drug linker (Formula II prepared as inExample 3) via a terminal maleimido moiety with a free sulfhydryl groupto create the disclosed SEZ6 ADC which is termed hSEZ6-1.ss1 ADC1herein. In addition, three control SEZ6 ADCs were fabricated byconjugating hSEZ6-1.ss1 to the same calicheamicin payloads butcomprising cleavable linkers (ADC2, ADC3 and ADC4). Finally, the controlADCs were made comprising hSEZ6-1.ss1 without the S6ON mutation.

The site-specific humanized SEZ6 ADC (hSEZ6-1.ss1) was conjugated usinga modified partial reduction process. The desired product is an ADC thatis maximally conjugated on the unpaired cysteine (C214) on each LCconstant region and that minimizes ADCs having a drug loading which isgreater than 2 while maximizing ADCs having a drug loading of 2. Inorder to further improve the specificity of the conjugation, theantibodies were selectively reduced using a process comprising astabilizing agent (e.g. L-arginine) and a mild reducing agent (e.g.glutathione) prior to conjugation with the linker drug, followed by adiafiltration and formulation step.

More specifically a preparation of each antibody was partially reducedin a buffer containing 1M L-arginine/5 mM EDTA with a pre-determinedconcentration of reduced glutathione (GSH), pH 8.0 for a minimum of 20hours at room temperature. All preparations were then buffer exchangedinto a 20 mM Tris/3.2 mM EDTA, pH 7.0 buffer using a 30 kDa membrane(Millipore Amicon Ultra) to remove the reducing buffer. The resultingpartially reduced preparations were then conjugated to the respectivecalicheamicin drug linker via a maleimide group for a minimum of 60mins. at room temperature. The pH was then adjusted to 6.0 with theaddition of 0.5 M acetic acid. Preparations of the ADCs were bufferexchanged into diafiltration buffer by diafiltration using a 30 kDamembrane. The dialfiltered SEZ6 ADC was then formulated with sucrose andpolysorbate-20 to the target final concentration.

The resulting formulation was then analyzed for protein concentration(by measuring UV), aggregation (SEC), drug to antibody ratio (DAR) byreverse-phase HPLC (RP-HPLC) and activity (in vitro cytotoxicity). Itwas then frozen and stored until use.

A schematic representation of hSEZ6-1.ss1 ADC1 is presented in FIG. 1appended hereto.

Example 5: In Vitro Characteristics of hSEZ6-1.Ss1 Antibody

Experiments were run to test whether the S6ON mutation affected theinteraction between the hSEZ6-1.ss1 mAb and the SEZ6 antigen. In thisregard the binding of soluble SEZ6-his antigen to surface-immobilizedSEZ6 antibodies and SEZ6 ADCs, with and without the S6ON mutation, weremeasured on a Biacore T200 (anti-human capture chip).

More specifically 5 μg/mL IgG were flowed for 12 sec at 5 μL/min,yielding 115-124 RU immobilization response. 22, 66 and 200 nM hSEZ6-hiswas injected for 90 sec at 30 μL/min, followed by 300 sec dissociation.Surfaces were regenerated by flowing 2M Magnesium Chloride (30 μL/min,30 sec) at the end of each cycle. Sensorgrams were double referenced(buffer injection and control flow cell) and are set forth in FIG. 2where the antibody with the mutation is labeled hSEZ6-1.ss1 and thesource antibody, without the mutation, is labeled hSEZ6-1.ss1 parent.

Additionally, affinity measurements were made using a Biacore T200 todetermine the binding characteristics of hSEZ6-1.ss1 comprising S60N. Inthis regard Fab constructs of the hSEZ6-1.ss1 antibody, with and withoutthe S60N mutation, were fabricated and purified. The binding of the Fabconstructs to soluble surface immobilized human SEZ6-His ligand was thencompared on a Biacore T200 (anti-His capture chip). More specifically of2 μg/mL human SEZ6-His were flowed across the chip for 12 sec at 5μL/min, yielding 45-66 RU immobilization response. 22, 66 and 200 nMinjections of Fab occurred for 90 sec at 30 μL/min, followed by 400-450second dissociation. Surfaces were regenerated by flowing 10 mM Glycine,pH 1.5 for 60 seconds at 30 uL/min. Sensorgrams were double referenced(buffer injection and control flow cell). The results are shown in Table4 immediately below.

TABLE 4 ka kd hSEZ6-His Rmax Analyte (1/Ms) (1/s) KD (nM) (RU) Wild TypeFab 1.1E+06 0.006 5.8 35.2 MutA (S60N) Fab 1.3E+06 0.006 4.8 4.0

A review of FIG. 2 shows that the S60N mutation and removal of theglycosylation site in the heavy chain variable region does notmaterially alter the binding properties of hSEZ6-1.ss1 antibodies whencompared with the parent antibody lacking the mutation. Similarly, theaffinity measurements shown in Table 4 demonstrate that the introducedmutation does not adversely impact the binding of the antibody used inthe disclosed ADC. As such, the potentially destabilizing position maybe modified without compromising the pharmaceutical effectiveness of themolecule.

Example 6: hSEZ6 ADCs Effectively Kill hSEZ6 Expressing Cells In Vitro

To determine whether anti-SEZ6 ADCs of the invention can efficientlymediate the delivery of conjugated cytotoxic agents to live cells, an invitro cell killing assay was performed using the anti-SEZ6 ADCs producedin Example 4 above.

Single cell suspensions of HEK293T cells overexpressing hSEZ6 or naïveHEK293T cells were plated at 500 cells per well into BD Tissue Cultureplates (BD Biosciences). One day later, various concentrations ofpurified ADC conjugated to calicheamicin were added to the cultures. Thecells were incubated for 96 hours. After the incubation viable cellswere enumerated using CellTiterGlo® (Promega) as per the manufacturer'sinstructions. Raw luminescence counts using cultures containingnon-treated cells were set as 100% reference values and all other countswere calculated as a percentage of the reference value.

FIG. 3 shows that all hSEZ6 expressing cells treated were much moresensitive to the anti-SEZ6 ADCs as compared to the naïve HET293T cells,demonstrating the specificity of the ADCs to the SEZ6 antigen.

The above results demonstrate the ability of anti-SEZ6 ADCs tospecifically mediate internalization and delivery of directly conjugatedcytotoxic payloads to cells expressing SEZ6.

Example 7: ADC Pharmacokinetics in Immunocompromised Mice

Pharmacokinetics (PK) of hSEZ6-1.ss1 ADC1, hSEZ6-1.ss1 ADC2, andhSEZ6-1.ss1 ADC3 were evaluated in NOD SCID mice. Mice (n=4 females pergroup) were randomized into treatment groups having equal average bodyweight, and then treated with the same amount of ADCs via a singleintravenous injection (100 μL volume). The ADCs were eachco-administered with 10 mg/kg unconjugated HuIgG1 antibody in order tosaturate the FcγR-mediated clearance and provide suitable ADC exposure.Serum samples were collected at 5 min, 4, 24, 72, 120, 168, 216, and 336hours after each dose, and total antibody (TAb) and ADC concentrationswere assessed by MSD immunoassay. Pharmacokinetics parameters includingmaximum concentrations (Cmax), exposure (area under the curve or AUC)evaluated from time=0 to 14 days post-dosing) and half-life, wereevaluated using non-compartmental analysis methods.

ADC and TAb serum pharmacokinetics declined bi-exponentially for all theADCs and peak concentrations (Cmax) were observed at 5 minutes postdose. There was no significant difference in ADC exposure (AUC 0-14Days) between the tested SEZ6 ADCs. ADC serum terminal half-life wassimilar between the ADCs. ADC stability was measured by the ratio of TAbto ADC exposures, and was similar for the tested compounds, ranging from1.4 to 1.6. Taken together, these data demonstrate that thepharmacokinetics of hSEZ6-1.ss1 ADC1, hSEZ6-1.ss1 ADC2, and hSEZ6-1.ss1ADC3 are comparable in NOD SCID mice.

Example 8: hSEZ6-1.ss1 Antibody Drug Conjugates Suppress Tumor Growth InVivo

In vivo experiments were conducted to confirm the cell killing abilityof the hSEZ6-1.ss1 ADC1, hSEZ6-1.ss1 ADC2, and hSEZ6-1.ss1 ADC3demonstrated in Example 6. To this end, site-specific SEZ6-targeted ADCsprepared as set forth in the previous Examples were tested for in vivotherapeutic effects in immunocompromised NOD SCID mice bearingsubcutaneous patient-derived xenograft (PDX) small cell lung cancer(SCLC) tumors having endogenous SEZ6 cell surface protein expression.Anti-SEZ6 conjugates hSEZ6-1.ss1 ADC1, hSEZ6-1.ss1 ADC2, and hSEZ6-1.ss1ADC3 were each tested in two different SCLC models.

SCLC-PDX lines, LU95 and LU149 were each injected as a dissociated cellinoculum under the skin near the mammary fat pad region and measuredweekly with calipers (ellipsoid volume=a×b²/2, where a is the longdiameter, and b is the short diameter of an ellipse). After tumors grewto an average size of 130-200 mm³ (range, 100-300 mm³) the mice wererandomized into treatment groups (n=5 mice per group) of equal tumorvolume averages. Mice (5 per group) were treated with identical singledoses of either vehicle (5% glucose in sterile water), or HuIgG1- orSEZ6-ADC preparations via an intraperitoneal injection (100 μL volume).SEZ6-ADC was co-administered with 10 mg/kg naked, HuIgG1 antibody inorder to linearize the pharmacokinetics.

Therapeutic effects assessed by weekly tumor volume (with calipers asabove) and weight measurements. Endpoint criteria for individual mice ortreatment groups included health assessment (any sign of sickness),weight loss (more than 20% weight loss from study start), and tumorburden (tumor volumes>1000 mm³). Efficacy was monitored by weekly tumorvolume measurements (mm³) until groups reached an average ofapproximately 800-1000 mm³. Tumor volumes were calculated as an averagewith standard error of the mean for all mice in treatment group and wereplotted versus time (days) since initial treatment. The results of thetreatments are depicted in FIGS. 4A and 4B where mean tumor volumes withstandard error of the mean (SEM) in 5 mice per treatment group areshown.

SEZ6-binding ADCs conjugated to calicheamicin (hSEZ6-1.ss1 ADC1,hSEZ6-1.ss1 ADC2, and hSEZ6-1.ss1 ADC3) were evaluated in mice bearingSCLC PDX-LU95 (FIG. 4A) or PDX-LU149 (FIG. 4B). Non-cleavable linkerADC1 had similar or greater efficacy compared to cleavable linker ADC2while cleavable linker ADC3 had greater efficacy compared to ADC1 at 2mg/kg. In any event, hSEZ6-1.ss1 ADC1 and hSEZ6-1.ss1 ADC3 can achievedurable responses for 50 days or longer in SCLC PDX. The response wasSEZ6-ADC specific, as there was no response observed following treatmentwith non-binding ADCs (HuIgG1) conjugated to the same calicheamicin druglinkers (data not shown).

Such results demonstrate that hSEZ6-1.ss1 ADC1, fabricated as set forthherein, has the potential to be pharmaceutically effective in retardingthe growth of small cell lung cancer cells.

Example 9: hSEZ6-1.ss1 ADC1 Exhibits a Robust Safety Margin

An analysis was conducted to determine the safety margin provided byhSEZ6-1.ss1 ADC1.

In this regard hSEZ6-1.ss1 ADC1, hSEZ6-1.ss1 ADC2 and hSEZ6-1.ss1 ADC3comprise an identical targeted mAb (hSEZ6-1.ss1) and warhead (N-acetylgamma calicheamicin), with differences in the linker drug attachment.hSEZ6-1.ss1 ADC1 is unique in that it comprises a non-cleavable linkeras compared to the other cathepsin B-susceptible di-peptide based linkerdrugs. The SEZ6 ADCs were evaluated in four discrete high-expressingSEZ6 SCLC mouse PDX models (LU64, LU86, LU95 and LU149), and in anexploratory repeat-dose toxicity study in cynomolgus monkeys.

A semi-mechanistic PK/PD model based on untreated tumor growth and SEZ6ADC-treated tumor response data in mouse was used to predict thetumor-static concentrations (TSC) that correspond to an ADCconcentration resulting in tumor stasis in patients. Subsequently, humanPK was simulated based on cynomolgus monkey PK data. Predictions ofhuman PK were then used to estimate the dose required to achieve aplasma trough concentration at the dose interval in patients that isequivalent to the TSC. Safety margins were estimated by comparing theADC exposures at the maximum tolerated dose (MTD) in cynomolgus monkeywith the exposure at the predicted human efficacious dose. This analysiswas repeated for each lung PDX models evaluated (LU64, LU86, LU95 andLU149).

Table 5 provides the predicted safety margin for each of the SEZ6 ADCs.Based on this analysis, hSEZ6-1.ss1 ADC1 was predicted to be moretolerable than the other SEZ6 ADCs (hSEZ6-1.ss1 ADC2, hSEZ6-1.ss1 ADC3)conjugated to the same payload but with cleavable linkers. This analysispredicted a safety margin of approximately 10 for hSEZ6-1.ss1 ADC1 whichis considerably higher than the two constructs with cleavable linkers.

TABLE 5 Compound Estimated Safety Margin hSEZ6-1.ss1 ADC1 10 hSEZ6-1.ss1ADC2 0.3 hSEZ6-1.ss1 ADC3 5.5

The predicted higher safety margin in humans based on data obtained incynomolgus monkeys, and corresponding dosing flexibility indicates thathSEZ6-1.ss1 ADC1 is a strong therapeutic candidate.

Example 10: hSEZ6-1.ss1 ADC1 is Particularly Active in SCLC

To further demonstrate the potential efficacy of the disclosed ADC,toxin specific assays were conducted using various tumor xenograft celllines. Initially SCLC, BR, CR, GA, NSCLC and PA PDX cell lines wereinterrogated via microarray analysis to determine the respectiveexpression level of SEZ6 antigen (FIG. 5A) and CD46, a known positivecontrol antigen (FIG. 5B). The microarray analysis was conducted usingthe Affymetrix ClariomD assay on purified RNA samples derived from humanPDX. A review of FIGS. 5A and 5B shows that, while SEZ6 expression isupregulated in SCLC tumors compared to other tumor types, the positiveantigen control CD46 exhibited consistently high mRNA expression levelsacross the panel of patient derived xenografts (PDX). Accordingly, theCD46 antigen was used as a surrogate ADC target to gauge the impact ofthe disclosed novel calicheamicin drug linker (Formula II) on varioustumor types.

In this respect N149, a humanized CD46 antibody (U.S. Pat. No.10,017,565 B2) was conjugated to the calicheamicin drug linker set forthherein or to a pyrrolobenzodiazepine (PBD) drug linker control. The ADCswere generated substantially as set forth in Example 4 above. Followingpreparation, the CD46 ADCs were frozen and stored until use.

PDX cells were inoculated into the flank of NOD-SCID mice. When tumorsreached between 100-300 mm3, PBD ADC preparations were introduced as asingle 1.6 mg/kg dose (FIG. 5C) while the calicheamicin ADC preparationswere administered as a single 8 mg/kg dose for all tumor types otherthan the SCLC PDX (FIG. 5D). For the SCLC PDX the calicheamicin ADCpreparation was administered as a 2 mg/kg or a 4 mg/kg dose (FIG. 5D).The tumors were then monitored for changes compared to non-targeting ADCpreparation with the same warhead. Delta Time to Tumor progression(dTTP) were calculated by subtracting the progression time fornontargeting ADC from the progression time for targeting agent. Tumorprogression was defined as the time pint where the observed measurementregrows at least 100 mm³ greater than the nadir volume post-treatment.Each data point in FIGS. 5C and 5D represents an individual PDX cellline of the respective tumor type.

As shown in FIG. 5C the PBD ADC preparations provided a relativelyuniform tumor response regardless of the PDX tumor type. In particular,susceptibility of the SCLC PDX to killing by the PBD toxin was largelyequivalent to that of the other tumor cell lines. In sharp contrast theSCLC PDX cell lines proved far more susceptible to killing by thecalicheamicin ADCs than the other PDX cell lines (FIG. 5D). Morespecifically, after a single, 8 mg/kg dose of the calicheamicin ADC themajority of patient derived xenografts from BR, CR, GA and NSCLC tumorsexhibited minimal to no response. There was a mixture of responses inpancreatic tumors but even the pancreatic tumors showed minimalresponses (<25 days dTTP) in the majority of cell lines tested.Conversely, the lower 2 mg/kg (black circles) or 4 mg/kg (white circles)doses of the calicheamicin ADC consistently reduced SCLC tumor growthsubstantially more than higher doses of the calicheamicin ADC achievedon other PDX tumors. Moreover, the majority of the SCLC tumors exhibitedgrowth delays of greater than 40 days as compared to a nontargetingantibody carrying the same warhead (data not shown).

These data suggest that SCLC tumors are more sensitive to acalicheamicin warhead than other DNA damaging warheads. This result wasunexpected, as the expectation was that the calicheamicin warhead wouldprovide similar results to those observed with the PBD warhead.

EMBODIMENTS

-   -   1. An isolated antibody that specifically binds human SEZ6        wherein the antibody comprises a heavy chain sequence of SEQ ID        NO:3 and a light chain sequence of SEQ ID NO:4.    -   2. The antibody of embodiment 1 wherein the antibody is        conjugated to a calicheamicin payload.    -   3. The antibody of embodiment 2 wherein the calicheamicin        payload comprises N-Ac calicheamicin.    -   4. The antibody of embodiment 3 wherein the calicheamicin        payload comprises Formula II.    -   5. A method of treating small cell lung cancer comprising        administering an antibody of any one of embodiments 1-4 to a        subject in need thereof    -   6. A kit comprising one or more containers containing an        antibody of any one of embodiments 1-4.    -   7. The kit of embodiment 6 further comprising a label or package        insert associated with the one or more containers indicating        that the antibody is for treating a subject having small cell        lung cancer.    -   8. A pharmaceutical composition comprising an antibody of any        one of embodiments 1-4.    -   9. A kit comprising one or more containers containing a        pharmaceutical composition of embodiment 8.    -   10. The kit of embodiment 9 further comprising a label or        package insert associated with the one or more containers        indicating that the pharmaceutical composition is for treating a        subject having small cell lung cancer.    -   11. A nucleic acid encoding all or part of an antibody of any        one of embodiments 1-4.    -   12. A vector comprising the nucleic acid of embodiment 11.    -   13. A host cell comprising the nucleic acid of claim 11 or the        vector of embodiments 12.    -   14. A SEZ6 ADC of the structure:

wherein Ab comprises an anti-SEZ6 antibody having a heavy chain sequenceof SEQ ID NO:3 and a light chain sequence of SEQ ID NO:4 and wherein nis 2.

-   -   15. A method of treating small cell lung cancer comprising        administering a SEZ6 ADC of embodiment 14 to a subject in need        thereof    -   16. A kit comprising one or more containers containing the SEZ6        ADC of embodiment 14.    -   17. The kit of embodiment 16 further comprising a label or        package insert associated with the one or more containers        indicating that the SEZ6 ADC is for treating a subject having        small cell lung cancer.    -   18. A pharmaceutical composition comprising the SEZ6 ADC of        embodiment 14.    -   19. The pharmaceutical composition of embodiment 18 wherein the        SEZ6 ADC of claim 14 is the predominant ADC species.    -   20. The pharmaceutical composition of embodiment 19 wherein the        predominant ADC species comprises greater than about 70% of the        ADC species present in the composition.    -   21. The pharmaceutical composition of embodiment 19 wherein the        predominant ADC species comprises greater than about 80% of the        ADC species present in the composition.    -   22. The pharmaceutical composition of embodiment 19 wherein the        predominant ADC species comprises greater than about 90% of the        ADC species present in the composition.    -   23. A kit comprising one or more containers containing any one        of the pharmaceutical compositions of embodiments 18-22.    -   24. The kit of embodiment 23 further comprising a label or        package insert associated with the one or more containers        indicating that the pharmaceutical composition is for treating a        subject having small cell lung cancer.    -   25. A method of treating small cell lung cancer comprising        administering any one of the pharmaceutical compositions of        embodiments 18-22.    -   26. A method of reducing tumor initiating cells in a tumor cell        population, wherein the method comprises contacting a tumor cell        population comprising tumor initiating cells and tumor cells        other than tumor initiating cells, with a SEZ6 ADC of embodiment        14 whereby the frequency of tumor initiating cells is reduced.    -   27. The method of embodiment 26, wherein the contacting is        performed in vivo.    -   28. The method of embodiment 26, wherein the contacting is        performed in vitro.    -   29. A method of delivering a cytotoxin to a cell comprising        contacting the cell with a SEZ6 ADC of embodiment 14.    -   30. A method of producing an ADC of embodiment 14 comprising the        step of conjugating a hSEZ6-1.ss1 antibody having a heavy chain        sequence of SEQ ID NO:3 and a light chain sequence of SEQ ID        NO:4 with a drug linker comprising Formula II.    -   31. The method of embodiment 30 further comprising the step of        lyophilizing the ADC.    -   32. A method of treating small cell lung cancer in a subject in        need thereof comprising administering a SEZ6 ADC having a safety        margin greater than 6 wherein the SEZ6 ADC comprises the        structure:

wherein Ab comprises an anti-SEZ6 antibody having a heavy chain sequenceof SEQ ID NO:3 and a light chain sequence of SEQ ID NO:4 and wherein nis 2.

-   -   33. The method of embodiment 32 wherein the safety margin is        about 10.    -   34. A calicheamicin drug linker, or a pharmaceutically        acceptable salt or solvate thereof, comprising the structure:

Those skilled in the art will further appreciate that the presentinvention may be embodied in other specific forms without departing fromthe spirit or central attributes thereof. In that the foregoingdescription of the present invention discloses only exemplaryembodiments thereof, it is to be understood that other variations arecontemplated as being within the scope of the present invention.Accordingly, the present invention is not limited to the particularembodiments that have been described in detail herein. Rather, referenceshould be made to the appended claims as indicative of the scope andcontent of the invention.

1.-19. (canceled)
 20. A calicheamicin drug linker, or a pharmaceuticallyacceptable salt or solvate thereof, comprising the structure: